Methods of using btl-ii proteins

ABSTRACT

The invention provides isolated BTL-II proteins, nucleic acids, antibodies, antagonists, and agonists and methods of making and using the same. Diagnostic, screening, and therapeutic methods using the compositions of the invention are provided. For example, the compositions of the invention can be used for diagnosis and treatment of inflammatory bowel diseases and for enhancing a mucosal immune response to an antigen.

This application is a divisional of U.S. patent application Ser. No.11/760,515, filed Jun. 8, 2007, now allowed, which is a divisional ofU.S. patent application Ser. No. 10/742,682, filed Dec. 19, 2003, nowU.S. Pat. No. 7,244,822, which claims benefit of U.S. ProvisionalApplication No. 60/436,185, filed Dec. 23, 2002 and U.S. ProvisionalApplication No. 60/525,298, filed Nov. 26, 2003, all of which areincorporated herein by reference.

FIELD OF THE INVENTION

The invention relates to butyrophilin-like proteins, specificallybutyrophilin-like proteins of the B7 subfamily, which are known tomodulate the function of immune effector cells such as, for example, Bcells and/or T cells. Nucleic acids encoding such proteins, processesfor producing such proteins, antibodies that bind to such proteins,pharmaceutical compositions containing such proteins or antibodies,methods of using such nucleic acids, proteins, and antibodies againstsuch proteins are also included.

BACKGROUND

Modulation of an immune or inflammatory response can be a valuable toolin controlling various kinds of diseases including autoimmune diseases,diseases characterized by abnormal inflammation and/or immune response,and infections. In treating diseases characterized by abnormalinflammation and/or immune responses, such as inflammatory boweldiseases and autoimmune or inflammatory diseases, down-modulation of animmune response is desirable. In other situations, for example whenvaccinating a patient to impart immunity to an infectious disease,stimulation of an immune response is desirable. In the vaccine setting,adjuvants that can heighten an immune response to a coadministeredantigen can be valuable in providing long term protection againstdisease. Particularly lacking in the art are adjuvants capable ofstimulating a mucosal immune response. A mucosal immune response, asopposed to a systemic immune response, is valuable because it can attackan infection at a very common point of entry, that is, at a mucosalsurface. The present invention addresses these needs in the art byproviding therapeutic agents to diagnose and treat diseasescharacterized by inappropriate and/or abnormal inflammation and/orimmune responses and therapeutic agents that can act as adjuvants tostimulate an immune response, particularly a mucosal immune response.

SUMMARY

The invention encompasses isolated BTL-II proteins, nucleic acids,antibodies, BTL-II inhibitors and agonists, and methods for using thesecompositions.

An isolated BTL-II protein comprising an amino acid sequence consistingof amino acids x-y of SEQ ID NO:4, wherein x is any amino acid fromposition 1 to 35 of SEQ ID NO:4 and y is any amino acid from position452-462 of SEQ ID NO:4 is provided. Such a BTL-II protein can compriseamino acids 30 to 453, 1 to 453, 29 to 457, 1 to 457, 1 to 482, and/or29 to 482 of SEQ ID NO:4. The invention further provides an isolatedBTL-II protein comprising a polypeptide consisting of an amino acidsequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, or98%, identical to amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14,or SEQ ID NO:18 or amino acids 126 to 156 of SEQ ID NO:16, wherein theidentity region of the amino acid sequence aligned with amino acids 127to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or amino acids 126to 156 of SEQ ID NO:16 is at least 20, optionally at least 25, or 30,amino acids long and the polypeptide can bind to a cell surface receptorexpressed on B cells or T cells and/or can inhibit the proliferation ofT cells. Such an amino acid sequence can be at least 150 amino acidslong and can be at least 80%, optionally at least 85%, 90%, 92%, 94%,96%, 98%, 99%, or 99.5%, identical to amino acids 30 to 358 of SEQ IDNO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18, wherein the identityregion of the amino acid sequence aligned with amino acids 30 to 358 ofSEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 is at least150, optionally at least 200, 250, or 300, amino acids. Further, theamino acid sequence can be at least 90%, optionally at least 92%, 94%,96%, 98%, 99%, or 99.5%, identical to amino acids 30 to 358 of SEQ IDNO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 and/or can compriseamino acids 30 to 358 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, orSEQ ID NO:18.

In another embodient the invention encompasses an isolated BTL-IIprotein comprising a first polypeptide consisting of a first amino acidsequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%,99%, or 99.5%, identical to amino acids 30 to 358 of SEQ ID NO:10, SEQID NO:14, SEQ ID NO:16, or SEQ ID NO:18, wherein the identity region ofthe first amino acid sequence aligned with amino acids 30 to 358 of SEQID NO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 is at least 150amino acids, wherein the first polypeptide comprises a secondpolypeptide consisting of a second amino acid sequence at least 80%,optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%,identical to amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, orSEQ ID NO:18, or amino acids 126 to 156 of SEQ ID NO:16, wherein theidentity region of the second amino acid sequence aligned with aminoacids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or aminoacids 126 to 156 of SEQ ID NO:16 is at least 20 amino acids long, andwherein the first polypeptide can inhibit the proliferation of T cells.The first amino acid sequence can be identical to amino acids 127 to 157of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18, or amino acids 126 to156 of SEQ ID NO:16,

Alternatively, the invention provides an isolated BTL-II proteincomprising a polypeptide consisting of an amino acid sequence at least80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%,identical to amino acids 30 to 457 of SEQ ID NO:4, wherein thepolypeptide comprises no more or less than 2 Ig-like domains, andwherein the polypeptide can inhibit the proliferation of T cells. Theamino acid sequence can be at least 80%, 85%, 90%, 92%, 94%, 96%, 98%,99%, 99.5%, or 100% identical to amino acids 30 to 247 of SEQ ID NO:8 orto amino acids 30 to 243 of SEQ ID NO:12. The amino acid sequence can beat least 90%, optionally at least 92%, 94%, 96%, 98%, 99%, 99.5%, or100%, identical to amino acids 30 to 457 of SEQ ID NO:4.

Alternatively, a BTL-II protein of the invention can comprise a firstpolypeptide consisting of a first amino acid sequence at least 80%,optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%,identical to amino acids 247 to 452 SEQ ID NO:4 or to amino acids 248 to447 of SEQ ID NO:6, wherein the first amino acid sequence does notcomprise an amino acid sequence at least 80%, optionally at least 85%,90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 32 to232 of SEQ ID NO:4 or to amino acid 27 to 232 of SEQ ID NO:6 with anidentity region of the first amino acid sequence aligned with SEQ IDNO:4 of at least 25, optionally, at least 50, 75, 100, or 150, aminoacids, and wherein the protein does not comprise a second polypeptideconsisting of a second amino acid sequence at least 80%, optionally atleast 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%, identical to aminoacids 32 to 232 of SEQ ID NO:4 or to amino acid 27 to 232 of SEQ ID NO:6with an identity region of the second amino acid sequence aligned withSEQ ID NO:4 of at least 25, optionally, at least 50, 75, 100, or 150,amino acids, and wherein the first polypeptide can inhibit theproliferation of T cells.

In another embodiment, an isolated BTL-II protein of the invention cancomprise a first polypeptide consisting of a first amino acid sequenceat least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or99.5%, identical to amino acids 32 to 242 of SEQ ID NO:8 or SEQ IDNO:12, wherein the identity region of the first amino acid sequencealigned with SEQ ID NO:8 or SEQ ID NO:12 is at least about 50,optionally at least about 75, 100, 150, or 200 amino acids long, whereinthe first polypeptide comprises a second polypeptide consisting of ansecond amino acid sequence at least 80%, optionally at least 85%, 90%,92%, 94%, 96%, 98%, 99%, or 99.5%, identical to amino acids 10 to 40 ofSEQ ID NO:8 or SEQ ID NO:12, wherein the identity region of the secondamino acid sequence aligned with SEQ ID NO:8 or SEQ ID NO:12 is at leastabout 20, optionally at least about 25 or 30, amino acids long, andwherein the first polypeptide can inhibit the proliferation of T cells.

In still another embodiment, the invention encompasses an isolatedBTL-II protein comprising a polypeptide consisting of an amino acidsequence at least 80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%,99%, or 99.5%, identical to amino acids 30 to 358 of SEQ ID NO:10, SEQID NO:14, SEQ ID NO:16, or SEQ ID NO:18, wherein the identity region ofthe amino acid sequence aligned with amino acids 30 to 358 of SEQ IDNO:10, SEQ ID NO:14, SEQ ID NO:16, or SEQ ID NO:18 is at least 250,optionally at least 275 or 300, amino acids, and wherein the polypeptidecan inhibit the proliferation of T cells.

BTL-II proteins of the invention can comprise a polypeptide that caninhibit the proliferation of T cells that may be at most about 480 aminoacids, about 380 amino acids, about 270 amino acids, or about 160 aminoacids in length.

The invention further encompasses an isolated BTL-II protein comprisinga first polypeptide consisting of a first amino acid sequence at least80%, optionally at least 85%, 90%, 92%, 94%, 96%, 98%, 99%, or 99.5%,identical to amino acids 32 to 358 of SEQ ID NO:10, wherein the identityregion of the first amino acid sequence aligned to SEQ ID NO:10 is atleast about 175, optionally about 200, 250, 275 or 300, amino acidslong, wherein the first amino acid sequence is not more than about 380,optionally not more than about 390, 270, or 170, amino acids in length,wherein the first polypeptide can inhibit the proliferation of T cells,wherein the first amino acid sequence is not at least 80% identical toamino acids 148 to 232 of SEQ ID NO:4 with an identity region of thefirst amino acid sequence aligned to amino acids 148 to 232 of SEQ IDNO:4 of at least about 20, 30, 40, 50, 60, or 75 amino acids, andwherein the BTL-II protein does not comprise a second amino acidsequence that is at least 80% identical to amino acids 148 to 232 of SEQID NO:4 with an identity region of the second amino acid sequencealigned to amino acids 148 to 232 of SEQ ID NO:4 of at least about 20,30, 40, 50, 60, or 75 amino acids. Such a BTL-II protein may compriseamino acids 32 to 242 of SEQ ID NO:8 or SEQ ID NO:12.

In another embodiment, the invention comprises a BTL-II recombinantfusion protein comprising the BTL-II protein and a heterologouspolypeptide, which can be an Fc region of an antibody or a leucinezipper. The invention also encompasses an immunogenic fragment of aminoacids 29 to 457 SEQ ID NO:4 that is capable of eliciting antibodies thatbind specifically to the fragment, that is at least 10 amino acids long,and that spans position 360 of SEQ ID NO:4. Immunogenic fragments of SEQID NO:10, SEQ ID NO:14, or SEQ ID NO:16, and SEQ ID NO:18 at least 10amino acids long are provided, wherein the immunogenic fragment spansposition 141 to 143 of SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:18, or SEQID NO:16 and can elicit antibodies that bind specifically to thefragment. Alternatively, the immunogenic fragments can span position 142of SEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or position 141 of SEQ IDNO:16.

In alternate embodiments, murine BTL-II proteins are provided.Specifically, the invention provides an isolated BTL-II proteincomprising an amino acid sequence consisting of amino acids x-y of SEQID NO:6, wherein x is any amino acid from position 1 to 35 of SEQ IDNO:6 and y is any amino acid from position 450-460 of SEQ ID NO:6. Sucha BTL-II protein can comprise amino acids 32-450, 29 to 456, and/or 29to 514 of SEQ ID NO:6.

Other embodiments include isolated antibodies that bind specifically toa BTL-II protein consisting of amino acids 1-457 of SEQ ID NO:4, 1-456of SEQ ID NO:6., 1-247 of SEQ ID NO:8, 1-363 of SEQ ID NO:10, 1-243 ofSEQ ID NO:12, 1-359 of SEQ ID NO:14, 1-358 of SEQ ID NO:16, or 1-362 ofSEQ ID NO:18. Such antibodies can be monoclonal antibodies, humanizedantibodies, or human antibodies and may inhibit the binding of BTL-II toits receptor. The invention encompasses nucleic acids that encode suchantibodies and cells that can produce such antibodies, which may behybridoma cells or cells that have been genetically engineered toproduce such an antibody. The invention further encompasses methods ofproducing antibodies by culturing such cells, which may secrete theantibody.

Other embodiments include BTL-II nucleic acids. The inventionencompasses an isolated BTL-II nucleic acid comprising a polynucleotideconsisting of nucleotides x to y of SEQ ID NO:3, wherein x is fromnucleotide 1 to 105 and y is from nucleotide 1345 to 1375, or comprisingthe complement of the polynucleotide. Such a nucleic acid can comprisenucleotides 105 to 1345 or 1 to 1371 of SEQ ID NO:3. Further, nucleicacids encoding immunogenic fragments are provided, as are BTL-II nucleicacids encoding any of the BTL-II proteins described above.

The invention further provides a vector comprising any of the BTL-IInucleic acids described above or nucleic acids encoding anti-BTL-IIantibodies and a host cell containing such a vector. Alternatively, theinvention provides a host cell genetically engineered to express aBTL-II protein, an immunogenic fragment of BTL-II, or an antibodyagainst BTL-II. Such host cells can be mammalian cells, including CHOcells. A method for producing a BTL-II protein, immunogenic fragment, oran anti-BTL-II antibody comprising culturing such a host cells underconditions allowing expression of the BTL-II protein, immunogenicfragment, or antibody is also encompassed by the invention. This methodmay further comprise isolating the BTL-II protein, immunogenic fragment,or antibody from the host cells or the medium. BTL-II proteins,immunogenic fragments, or antibodies produced by such methods are alsocontemplated. The invention further encompasses mammalian cells thatproduce antibodies against BTL-II, including hybridoma or myeloma cellsand methods for making antibodies by culturing such cells.

Various therapeutic methods employing the compositions encompassed bythe invention are also contemplated. The invention provides a method forreducing inflammation in the gut in a patient suffering from aninflammatory bowel disease, optionally either Crohn's disease orulcerative colitis, comprising administering a therapeutically effectiveamount of a BTL-II protein, optionally a soluble BTL-II protein. Amethod for inducing an immune response, including a system and/or amucosal immune response, against an antigen comprising administering atherapeutically effective amount of a BTL-II antagonist and the antigenis also provided. The BTL-II antagonist can be an antibody or a smallmolecule, and the antigen can be administered directly to a mucosalsurface or can be administered systemically. Further provided is amethod for diagnosing an inflammatory bowel disease or predicting theonset of an inflammatory bowel disease comprising assaying a tissuesample from the bowel of a patient to determine whether BTL-II mRNA orprotein is overexpressed. The tissue can be assayed for BTL-II proteinexpression using an anti-BTL-II antibody. The invention further providesa method for dampening an immune response to an antigen, especially anauto-antigen in a patient suffering from an autoimmune or inflammatorydisease, comprising co-administering the antigen and a soluble BTL-IIprotein. The antigen can be administered via a mucosal surface.

In further embodiments, the invention encompasses methods for inhibitingT cell proliferation and cytokine production. In one embodiment, theinvention comprises a method for inhibiting T cell proliferationcomprising contacting the T cells with a BTL-II polypeptide. The T cellscan be human T cells and can be contacted with the BTL-II polypeptide invivo. As an alternative to a BTL-II polypeptide, an agonistic antibodythat binds to a BTL-II receptor expressed on T cells can be used,provided that it can inhibit the proliferation of T cells. In anotherembodiment, the invention includes a method for suppressing cytokineproduction by a T cell comprising contacting the T cell with a BTL-IIpolypeptide. The T cells can be human T cells and can be contacted withthe BTL-II polypeptide in vivo. The cytokine can be, for example,interferon gamma (IFNγ), interleukin 2(IL2), or interleukin 5 (IL5).

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the domain structures of selected members of the family ofbutyrophilin-like proteins. Most of the selected proteins are members ofthe B7 subfamily discussed below. The name of each protein is shown tothe left of the diagram depicting its structure. The “Ig-like” domainsare immunoglobulin-like domains as described below. The domains labeled“7” are heptad repeat regions as described below. The domains labeled“TM” are transmembrane domains. Open boxes are cytoplasmic domains notidentified are part of a specific family of domains. Domains labeled“B30.2” are B30.2 domains as explained below. The BTL-II protein isdepicted here as having four Ig-like domains, but forms of BTL-II alsoexist that have two or three Ig-like domains. B7-H3 is depicted withfour Ig-like domains, and it also exists in a form containing only twoIg-like domains.

FIG. 2 is a diagram of the structure the human BTL-II gene and mRNA. Theboxes indicate exons. The numbers below the horizontal lines below theboxes indicate the positions within SEQ ID NO:3 of the exons. Thehorizontal lines at the bottom of the figure denote the extent of SEQ IDNO:3 encoding the extracellular domain and the transmembrane andcytoplasmic domains (“TM/Cyto”) and forming the 3′ untranslated region(“3′ UTR”). Stop codons are denoted by a mark that could be described asa sunburst or a small explosion.

FIG. 3 a is a diagram of the structure of a first category of splicevariants of the human BTL-II mRNA. Symbols are the same as described forFIG. 2. The large Xs over exons 2 and 3 indicate that these exons aremissing in this category of splice variants.

FIG. 3 b is a representative sequence of a member of the first categoryof splice variants (bottom line, SEQ ID NO:7) aligned to a portion ofSEQ ID NO:3 (top line). There are no mismatches other than the gapcreated by the missing exons 2 and 3.

FIG. 4 a shows the structure of a second category of splice variants ofthe human BTL-II RNA. Symbols are the same as described for FIG. 2. Thelarge X over exon 3 indicates that this exon is missing in this categoryof splice variants.

FIG. 4 b shows a representative sequence of a member of the secondcategory of splice variants (bottom line, SEQ ID NO:9) aligned to aportion of SEQ ID NO:3 (top line). There are no mismatches other thanthe gap created by the missing exon 3.

FIG. 5 a shows the structure of a third category of splice variants ofthe human BTL-II mRNA. Symbols are the same as described for FIG. 3. Thelarge Xs over exons 2 and 3 indicate that these exons are missing inthis category of splice variants. The stars accompanied by numbersadjacent to the boxes indicate the positions of sequence polymorphismspresent in this category of splice variants. The variations are presentat the following positions within SEQ ID NO:3: variation 2 is atposition 1050; variation 3 is at positions 1136 and 1140; variation 4 isat positions 1178 and 1179; and variation 5 is at position 1212; andvariation 6 is at position 1242.

FIG. 5 b shows a representative sequence of a member of the thirdcategory of splice variants (bottom line, SEQ ID NO:11) aligned to aportion of SEQ ID NO:3 (top line). Mismatched bases are indicated inboldface.

FIG. 6 a shows the structure of a fourth category of splice variants ofthe human BTL-II mRNA. The large X over exon 3 indicates that this exonis missing in this category of splice variants. Sequence polymorphismsare indicated as in FIG. 5 a, and variations 2 to 6 are as in FIG. 5 a.

FIG. 6 b shows a representative sequence of a member of the fourthcategory of splice variants (bottom line, SEQ ID NO:13) aligned to aportion of SEQ ID NO:3 (top line). Mismatched bases are indicated inboldface.

FIG. 7 a shows the structure on a fifth category of splice variants ofthe human BTL-II mRNA. The large X over exon 3 indicates that this exonis missing in this category of splice variants. Sequence polymorphismsare indicated as in FIG. 5 a, and variations 2 to 6 are as in FIG. 5 a.Variation 1 is a deletion of nucleotides 78 to 80 of SEQ ID NO:3.

FIG. 7 b shows a representative sequence of a member of the fifthcategory of splice variants (bottom line, SEQ ID NO:15) aligned to aportion of SEQ ID NO:3 (top line). Mismatched bases are indicated inboldface.

FIG. 8 is a diagram of the structure of the murine BTL-II gene and mRNA.Symbols are as in FIG. 5 except that the number refer to positions inSEQ ID NO:5.

FIG. 9 a shows the structure of a first category of splice variants ofthe murine BTL-II mRNA. The large X over exon 3 indicates that this exonis missing in this category of splice variants.

FIG. 9 b shows a representative sequence of a member of the firstcategory of murine splice variants (bottom line, SEQ ID NO:17) alignedto SEQ ID NO:5 (top line). There are no mismatches other than the gapcreated by the missing exon 3.

FIG. 10 represents expression of BTL-II mRNA in colonic tissue ofmdr1a−/−mice (which carry the mdr1a null mutation in an FVB background)when exhibiting no symptoms of inflammatory bowel disease (horizontalstripes) or when exhibiting symptoms of inflammatory bowel disease(checkerboard pattern) relative to expression of BTL-II mRNA in colonictissue of non-symptomatic, wild type FVB mice. Measurements were done byhybridizing fluorescently-labeled cDNA to an Affymetrix chip containingan oligonucleotide complementary to BTL-II mRNA.

FIG. 11 is a graph showing the relative concentrations of BTL-II mRNAdetected by a real time PCR assay of colon tissue from two differentwild type mice of the FVB strain (FVB #1 and #2; diagonal lines) andfrom two mdr1a−/−mice showing symptoms of inflammatory bowel disease(Mdr1a−/−#1 and #2; checkerboard patterns).

FIG. 12 a is a bar graph showing proliferation (as evidenced by uptakeof ³H-thymidine) of purified human T cells cultured with the followingproteins: anti-CD3ε antibody alone,

; anti-CD3ε antibody and BTL-II:Fc,

; anti-CD3ε antibody and B7RP-1:Fc,

; anti-CD3ε antibody, B7RP-1:Fc, and BTL-II:Fc,

; anti-CD3ε antibody and B7-2:Fc,

; and anti-CD3ε antibody, B7-2:Fc, and BTL-II:Fc,

.

FIG. 12 b is identical to FIG. 12 a, except that a linear, rather than alogarithmic scale is used.

FIG. 13 is a bar graph showing proliferation of purified human T cellsin response to a constant amount of anti-CD3ε antibody and a varyingamount of BTL-II:Fc, as indicated.

FIG. 14 is a bar graph showing proliferation of murine T cells inresponse to anti-CD3ε antibody alone,

, anti-CD3ε antibody plus BTL-II:Fc,

, or anti-CD3ε antibody plus a control protein consisting of a human Fcregion,

.

FIG. 15 is a bar graph showing proliferation of murine B cells inresponse to no added protein,

, TALL-1 protein alone,

, an anti-IgM antibody alone,

, TALL-1 plus and anti-IgM antibody,

, and TALL-1, anti-IgM antibody, and BTL-II:Fc,

.

FIG. 16 a shows proliferation of purified human T cells in response tovarious combinations of proteins indicated as in FIG. 12 a.

FIG. 16 b shows the relative interferon gamma (IFNγ) production inresponse to various combinations of proteins indicated as in FIG. 12 a.

FIG. 16 c shows the relative interleukin 2 (IL2) production in responseto various combinations of proteins indicated as in FIG. 12 a.

FIG. 16 d shows the relative interleukin 5 (IL5) production in responseto various combinations of proteins indicated as in FIG. 12 a.

FIG. 17 is a bar graph showing the total number of dead cells incultures of purified T cells cultured with various combinations ofproteins, as indicated in FIG. 12 a.

FIG. 18 shows FACS scans of cells transfected with either an emptyvector (top line), or a vector containing cDNA encoding murine BTL-II(second line), murine B7RP-1 (third line), or murine CD80 (bottom line).The cells were stained with the following proteins: (1) theextracellular region of murine CTLA4 fused to a human Fc region of anIgG1 antibody (first column); (2) the extracellular region of murineCD28 fused to a human Fc region from an IgG1 antibody (second column);(3) the extracellular region of murine ICOS fused to a human Fc regionfrom an IgG1 antibody (third column); and (4) the extracellular regionof PD1 fused to a human Fc region of an IgG1 antibody (fourth column).The vertical axis of each scan (labeled “counts”) represents cell numberand the horizontal axis (labeled “FL2-H”) represents fluorescence. Thehorizontal line labeled “M1” shows where the “gate” was set on the FACSmachine. Cells encompassed in this gate are considered positive; allothers are considered negative. The small lettering above each FACS scanindicates an individual sample number.

BRIEF DESCRIPTION OF THE SEQUENCE LISTINGS

SEQ ID NO:1 is the nucleotide sequence of the human BTL-II cDNA from theNational Center for Biotechnology Information (NCBI) entry with theaccession number NM_(—)019602.

SEQ ID NO:2 is the amino acid sequence of the of the human BTL-IIprotein predicted from the cDNA sequence of the NCBI entry with theaccession number NM_(—)019602.

SEQ ID NO:3 is the nucleotide sequence of a full length human BTL-IIcDNA of the invention.

SEQ ID NO:4 is the amino acid sequence of the full length human BTL-IIprotein encoded by SEQ ID NO:3.

SEQ ID NO:5 is the nucleotide sequence of the full length murine BTL-IIcDNA of the invention.

SEQ ID NO:6 is the amino acid sequence of the full length murine BTL-IIprotein encoded by SEQ ID NO:5.

SEQ ID NO:7 is the nucleotide sequence of the cDNA from a representativemember of the first category of human BTL-II splice variants (FIG. 3 a).

SEQ ID NO:8 is the amino acid sequence encoded by SEQ ID NO:7.

SEQ ID NO:9 is the nucleotide sequence of the cDNA from a representativemember of the second category of human BTL-II splice variants (FIG. 4a).

SEQ ID NO:10 is the amino acid sequence encoded by SEQ ID NO:9.

SEQ ID NO:11 is a partial nucleotide sequence of the cDNA from arepresentative member of the third category of human BTL-II splicevariants (FIG. 5 a).

SEQ ID NO:12 is the amino acid sequence encoded by SEQ ID NO:11.

SEQ ID NO:13 is a partial nucleotide sequence of the cDNA from arepresentative member of the fourth category of human BTL-II splicevariants (FIG. 6 a).

SEQ ID NO:14 is the amino acid sequence encoded by SEQ ID NO:13.

SEQ ID NO:15 is a partial nucleotide sequence of the cDNA from arepresentative member of a fifth category of human BTL-II splicevariants (FIG. 7 a).

SEQ ID NO:16 is the amino acid sequence encoded by SEQ ID NO:15.

SEQ ID NO:17 is the nucleotide sequence of a representative member of afirst category of murine BTL-II splice variants (FIG. 9 a).

SEQ ID NO:18 is the amino acid sequence encoded by SEQ ID NO:17.

SEQ ID NO:19 is the nucleotide sequence encoding the BTL-II:Fc fusionprotein described in Example 5.

SEQ ID NO:20 is the amino acid sequence of the BTL-II:Fc fusion proteindescribed in Example 5.

DETAILED DESCRIPTION

The present invention provides BTL-II proteins and nucleic acids,including recombinant vectors encoding BTL-II proteins, anti-BTL-IIantibodies, which can be agonists or antagonists, as well as methods forproducing and using these molecules and pharmaceutical compositionscontaining them. BTL-II expression is restricted to a small number oftissue types. BTL-II is overexpressed in the gut prior to the onset ofsymptoms and during the symptomatic phase in a murine inflammatory boweldisease model system as illustrated in Example 4. BTL-II antibodies cantherefore serve to diagnose or to predict the likelihood of the onset ofan inflammatory bowel disease. In addition, the invention provides anumber of allelic variants of the BTL-II nucleotide sequence (FIGS. 5 ato 7 a). These can find use in predicting susceptibility to inflammatorybowel disease. Further, since a soluble BTL-II protein can inhibit Tcell proliferation and cytokine production (Examples 6-10), BTL-IIproteins can find use in the treatment of autoimmune and inflammatorydiseases.

Further, BTL-II is expressed in Peyer's patches, which are specializedstructures known to play a role in immune sampling in the gut. BTL-II ispreferentially expressed on are CD11c⁺ (low expressing) CD8⁺ B220⁺dendritic cells (also called plasmacytoid dendritic cells) found inPeyer's patches as compared to other cells found in Peyer's patches,including other dendritic cells. Peyer's patch dendritic cells have beenhypothesized to play a role in inducing tolerance at mucosal surfacesdue to their influence on T cell differentiation. Weiner (2001), NatureImmunology 2(8): 671-71; Weiner (2001), Immunol. Rev. 182: 207-14;Iwasaki and Kelsall (1999), American Journal ofPhysiology-Gastrointestinal and Liver Physiology 276(5): G1074-78. Thus,anti-BTL-II antibodies can be used to identify CD11c⁺ (low expressing)CD8⁻ B220⁺ dendritic cells within Peyer's patches.

As explained below, BTL-II is within the B7 subfamily ofbutyrophilin-like proteins that play roles in regulating T and Bcell-mediated responses. BTL-II may play a role in either dampeningand/or promoting immune system-mediated inflammation, especially in thegut. Given the complexity of the immune system, a single cell surfaceprotein may, in some cases, both stimulate or dampen an immune responseby immune effector cells, depending on what receptors on the effectorcells are available for the molecule to interact with. The B7-1 and B7-2proteins discussed below are examples of immune-regulating cell surfaceproteins with both stimulatory and dampening effects on immune effectorcells, in this case, T cells. Hence, BTL-II may play similar dual rolesin vivo. However, the gut is, overall, highly tolerant to foreignantigens, as evidenced by its tolerance to food antigens and commensalmicroorganisms. It is therefore likely that BTL-II plays a role indampening immune responses or inflammation in vivo in at least somesituations. Thus, BTL-II antagonists, which can include antibodies,binding proteins selected in vitro, or small molecules, may serve tostimulate a mucosal immune response to an antigen. Further, solubleBTL-II proteins, or functionally equivalent anti-idiotypic antibodies,may dampen an immune response in the gut or in other mucosal surfaces inthe body, such as the lungs.

An “antibody,” as used herein, can be a chimeric antibody, can bemonomeric or single chain, dimeric, trimeric, tetrameric, or multimericantibody, and can be a recombinant protein or a non-recombinant protein.A “domain” is part or all of a protein that can be distinguished byprimary sequence motifs and/or tertiary structural characteristics.Programs designed to locate protein domains include, for example, Pfam(Bateman et al. (1999), Nucleic Acids Res. 27: 260-62; Bateman et al.(1999), Nucleic Acids Res. 28: 263-66), ProDom (Corpet et al. (1999),Nucleic Acids Res. 27: 263-67; Corpet et al. (1999), Nucleic Acids Res.28: 267-69), Domo (Gracy and Argos (1998), Bioinformatics 14: 164-87),and SMART (Ponting et al. (1999), Nucleic Acids Res. 27: 229-32).Tertiary structure can be determined empirically, for example by X-raycrystallography, or can be predicted using computer software designedfor such uses. For example, structural data can be accessed through theEntrez website of NCBI from the Molecular Modeling Database (Wang et al.(2000), Nucleic Acids Res. 28(1): 243-45) or by the use of software suchas DALI (Holm and Sander (1993), J. Mol. Biol. 233: 123-38).Immunoglobulin-like domains (Ig-like), for example, are distinguishedmainly by their tertiary structure rather than by primary sequencehomologies. See e.g. Bork et al. (1994), J. Mol. Biol. 242: 309-20;Hunkapiller and Hood (1989), Adv. Immunol. 44: 1-63; Williams andBarclay (1988), Ann. Rev. Immunol. 6: 381-405. However, IgV and IgCdomains do contain a handful of highly conserved amino acids that occurat conserved positions within their primary amino acid sequence. Seee.g. Kabat et al. (1991), Sequences of Proteins of ImmunologicalInterest, U.S. Dept. of Health and Human Services, Public HealthService, National Institutes of Health, NIH Publication No. 91-3242. Thepresence of such highly conserved amino acids occurring in the properspacing can indicate the presence of an IgC-like or IgV-like domain.

A nucleic acid “encodes” a protein, as meant herein, if the nucleic acidor its complement comprises the codons encoding the protein.

Cells have been “genetically engineered” to express a specific proteinwhen recombinant nucleic acid sequences that allow expression of theprotein have been introduced into the cells using methods of “geneticengineering,” such as viral infection, transfection, transformation, orelectroporation. See e.g. Kaufman et al. (1990), Meth. Enzymol. 185:487-511. This can include, for example, the introduction of nucleicacids encoding the protein into the cells or the introduction ofregulatory sequences to enhance the expression of a host gene encodingthe protein as described in U.S. Pat. No. 5,272,071 to Chappel. Themethods of “genetic engineering” encompass numerous methods including,but not limited to, amplifying nucleic acids using polymerase chainreaction, assembling recombinant DNA molecules by cloning them inEscherichia coli, restriction enzyme digestion of nucleic acids,ligation of nucleic acids, in vitro synthesis of nucleic acids, andtransfer of bases to the ends of nucleic acids, among numerous othermethods that are well-known in the art. See e.g. Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2^(nd) ed., vol. 1-3, ColdSpring Harbor Laboratory, 1989.

A “heterologous polypeptide” is any polypeptide that is at least 3 aminoacids long that is not a BTL-II polypeptide as meant herein.

In connection with comparisons to determine sequence identity ofpolynucleotides or polypeptides, what is meant by an “identity region”is the portion of the polynucleotide or polypeptide that is matched,partially or exactly, with another polynucleotide or polypeptide by thecomputer program GAP (Devereux et al. (1984), Nucleic Acids Res. 12:387-95) using the parameters stated below. For example, when apolypeptide of 20 amino acids is aligned with a considerably longerprotein, the first 10 amino acids match the longer protein exactly, andthe last 10 amino acids do not match the longer protein at all, theidentity region is 10 amino acids. If, on the other hand, the first andlast amino acids of the 20 amino acid polypeptide match the longerprotein, and eight other matches are scattered between, the identityregion is 20 amino acids long. However, long stretches in either alignedstrand without identical or conservatively substituted amino acids oridentical nucleotides of at least, for example, 20 amino acids or 60nucleotides constitute an endpoint of an identity region, as meantherein.

“Ig-like” domains are immunoglobulin like domains and may be eitherIgV-like or IgC-like domains or be domains that can fold into animmunoglobulin structure but cannot be unambigously classified as eitherIgV-like or IgC-like.

“IgV-like” domains have amino acid sequences that can be folded into animmunoglobulin fold with the characteristics common to immunoglobulinvariable region domains. See Bork et al., supra; Miller et al. (1991),Proc. Natl. Acad. Sci. USA 88: 4377-81; Williams and Barclay (1988),Ann. Rev. Immunol. 6: 381-405. One of skill in the art is aware that thepresence of amino acids that are highly concserved in IgV domains atconserved positions can identify a domain as IgV-like.

“IgC-like” domains have amino acid sequences that can be folded into animmunoglobulin fold with the characteristics common to immunoglobulinconstant region domains. See Bork et al., supra; Williams and Barclay,supra. One of skill in the art is aware that the presence of amino acidsthat are highly concserved in IgC domains at conserved positions canidentify a domain as IgC-like.

“Inflammatory bowel diseases” include Crohn's disease, ulcerativecolitis, ileitis, and any other disease characterized by chronicinflammation of the gastrointestinal tract.

A protein comprises “no more or less than 2 Ig-like domains” when itcontains two Ig-like domains and does not contain all or somerecognizable portion of another Ig-like domain. However, such a proteincan contain other amino acid sequences that are not Ig-like domains andstill contain “no more or less than 2 Ig-like domains.” Thus, the phrase“no more or less” refers only to Ig-like domains, not to other aminoacid sequences that may be part of the protein.

When a polypeptide is said to be able to “inhibit the proliferation of Tcells” or to be able to perform some other biological function, it ismeant that a protein comprising the polypeptide can perform the functionand that the addition of the polypeptide to at least some proteins thatcannot perform the biological function enables these proteins to performthe function. In some cases, a polypeptide that can perform thebiological function can do so without any additional sequences. In othercases, a polypeptide may require other sequences, for example,oligomerization sequences, to perform a biological function. In onescenario, a polypeptide may be able to effectively perform a particularbiological function when it is linked to an Fc region or a leucinezipper or some other dimerizing domain, but not without the dimerizingdomain. As meant herein, such a polypeptide can perform the biologicalfunction.

A “protein” is any polypeptide comprising at least 10 amino acids,optionally at least 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, and/or300 amino acids.

“Recombinant,” as it applies to polypeptides or proteins, means that theproduction of the protein is dependent on at least one step in whichnucleic acids, which may or may not encode the protein, are introducedinto a cell in which they are not naturally found.

“Recombinant fusion proteins” are recombinant proteins comprising partor all of at least two proteins, which are not found fused together innature, fused into a single polypeptide chain. A “silent mutation” in anucleic acid sequence is one that changes the sequence of the nucleicacid without changing the sequence of the protein encoded by the nucleicacid.

A “soluble” protein is one lacking a transmembrane domain or some otheramino acid sequence, such as a GPI anchor sequence, that normally causesthe protein to be embedded in or to associate with a membrane. Suchproteins might typically comprise all or part of the extracellularregion of a transmembrane protein.

For the purposes of the invention, two proteins or nucleic acids are“substantially similar” if they are at least 80%, optionally at least85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% identical to each other inamino acid or nucleotide sequence and maintain or alter in a desirablemanner a biological activity of the unaltered protein. The percentidentity of two amino acid or two nucleic acid sequences can bedetermined by visual inspection and mathematical calculation, or morepreferably, the comparison is done by comparing sequence informationusing a computer program. An exemplary computer program is the GeneticsComputer Group (GCG; Madison, Wis.) Wisconsin package version 10.0program, GAP (Devereux et al. (1984), Nucleic Acids Res. 12: 387-95).The preferred default parameters for the GAP program includes: (1) TheGCG implementation of a unary comparison matrix (containing a value of 1for identities and 0 for non-identities) for nucleotides, and theweighted amino acid comparison matrix of Gribskov and Burgess, ((1986)Nucleic Acids Res. 14: 6745) as described in Atlas of PolypeptideSequence and Structure, Schwartz and Dayhoff, eds., National BiomedicalResearch Foundation, pp. 353-358 (1979) or other comparable comparisonmatrices; (2) a penalty of 8 for each gap and an additional penalty of 2for each symbol in each gap for amino acid sequences, or a penalty of 50for each gap and an additional penalty of 3 for each symbol in each gapfor nucleotide sequences; (3) no penalty for end gaps; and (4) nomaximum penalty for long gaps. Other programs used by those skilled inthe art of sequence comparison can also be used.

Butyrophilin-Like Proteins

Butyrophilin-like proteins reported to date share some structuralfeatures, and many are encoded within or adjoining the majorhistocompatability locus (MHC). See e.g. Henry et al. (1999), ImmunologyToday 20(6): 285-88. Butyrophilin is a protein that constitutes 40% ofthe total protein associated with the fat globule of bovine milk, and ahuman homolog exists. Ruddy et al. (1997), Genome Research 7:441-56,citing Jack and Mather (1990), J. Biol. Chem. 265: 14481-86. Similarityat the amino acid sequence level among butyrophilin-like proteins can below, but domain structure is somewhat conserved. All members comprise anamino terminal signal peptide followed by an Ig-like domain, usuallyreported to be an IgV-like domain. In many cases, this is followed byanother Ig-like domain, usually reported to be an IgC-like domain, atransmembrane domain, and a cytoplasmic domain. The two Ig-like domainscan be repeated immediately following their first occurrence, as inBTL-II or B7-H3. This domain structure is illustrated in FIG. 1. Most ofthe proteins diagrammed in FIG. 1 are members of the B7 subfamily ofbutyrophilin-like proteins; only three (human BTN2A1, humanbutyrophilin, and myelin oligodendrocyte glycoprotein) are not.

Ig-like domains can be very divergent in sequence and still retain oneof a number of conserved folding patterns, which all include a commonstructural core comprising four β strands. Immunoglobulin constant andvariable regions have a tertiary structure which is characterized byseven to nine antiparallel β strands forming a barrel-like shape. Borket al. (1994), J. Mol. Biol. 242: 309-20. IgV- and IgC-likeimmunoglobulin domains each include a handful of distinct, highlyconserved residues. Hunkapiller and Hood (1989), Adv. Immunol. 44: 1-63;Miller et al. (1991), Proc. Natl. Acad. Sci. USA 88: 4377-81; Williamsand Barclay (1988), Ann. Rev. Immunol. 6: 381-405. Conserved residuesare presumably important for structure or function. Such conservedresidues are found in the Ig-like domains of BTL-II. For example, thefirst Ig-like domain of the human BTL-II protein (SEQ ID NO:4) containssuch highly conserved residues characteristic of IgV-like domains in theappropriate locations at, for example, positions G43, C50, W65, L109,D118, G120, Y122 and C124. See Table 3. The third Ig-like domain alsocontains residues that correspond to residues conserved in IgV-likedomains. The second and fourth Ig-like domains of human BTL-II containcontain residues that correspond to highly conserved residues inIgC-like domains. Table 3.

Transmembrane and cytoplasmic domains occur in butyrophilin-likeproteins, regardless of whether they have a second Ig-like domain. Thetransmembrane domain may or may not be followed by one or more sevenamino acid units reminiscent of the heptad repeats typical of anα-helical coiled coil motif. Heptad repeats may also occur at otherpositions in a butyrophilin-like protein. For a discussion of heptadrepeats, see Miller et al. (1991), Proc. Natl. Acad. Sci. 88: 4377-81.Methods for predicting transmembrane domains are well known in the art.See e.g. Ikeda et al. (2002), In Silico Biol. 2(1): 19-33; Tusnady andSimon (1998), J. Mol. Biol. 283(2):489-506. Butyrophilin-like proteinscan have a cytoplasmic domain. Such a cytoplasmic domain may or may notcomprise a B30.2 domain. Sequences of various B30.2 domains aredisplayed, and putative functions of B30.2 domains are discussed byHenry et al. ((1998), Mol. Biol. Evol. 15(12): 1696-1705. B30.2 domainsare found in a variety of proteins, and the function of the B30.2 domainis unknown. A proposed ligand of the B30.2 domain is xanthine oxidase,which interacts with the cytoplasmic domain of butyrophilin. Mutationsin B30.2 domains of two different B30.2-containing proteins have beencorrelated with two different diseases, although causal relationshipsbetween the mutations and the disease phenotypes have not beenestablished. Henry et al., supra.

The B7 Subfamily of Butyrophilin-Like Proteins

BTL-II shares a domain structure with a number of butyrophilin-likeimmune regulatory proteins lacking the B30.2 domain that play roles inregulating immune effector cells, such as, for example, T cells, B cellsand myeloid cells. This subfamily is referred to herein as “the B7subfamily” of butyrophilin-like proteins. As meant herein,characteristics of B7 subfamily members include, without limitation, thefollowing: (1) having one or more extracellular Ig-like domains; (2)having transmembrane and cytoplasmic domains; (3) lacking a B30.2domain; (4) being expressed on antigen presenting cells; (5) undergoingregulation of expression during an activated immune response; and (6)modulating an immune response. No secreted, soluble B7 proteins, lackinga transmembrane domain, that modulate immune response have been reportedto date. Most B7 proteins have two extracellular Ig-like domains, butsome isoforms of B7-H3 have four. See FIG. 1. Human BTL-II can have fromtwo to four extracellular Ig-like domains and has all of thecharacteristics of B7 family members listed above. We therefore considerit to be a member of the B7 subfamily of butyrophilin-like proteins. Allother known members of the B7 subfamily interact with receptors onimmune effector cells, such as B cells and/or T cells, which interactionserves as a signal to modulate an immune response. BTL-II is thereforepredicted to interact with a receptor on an immune effector cell, suchas a T cell, and thereby to modulate an immune response.

B7 family members play roles in modulating the activity of immuneeffector cells, especially T cells. Henry et al. (1999), ImmunologyToday 20(6): 285-88; Sharpe and Freeman (2002), Nat. Rev. Immunol. 2:116-26. The best characterized examples are, CD80 and CD86 (also calledB7-1 and B7-2, respectively), which are expressed on antigen presentingcells and can promote T cell activation when they interact with CD28,which is constitutively expressed on the surface of T cells, or inhibitT cell activation when they interact with a CTLA-4, which is alsoexpressed on the surface of T cells. CTLA-4 expression is notconstitutive, but is rapidly upregulated following T cell activation.See e.g. Masteller et al. (2000), J. Immunol. 164: 5319-27; Hehner etal. (2000), J. Biol. Chem. 275(24): 18160-71; Sharpe and Freeman (2002),Nat. Rev. Immunol. 2: 116-26. Moreover, it has been reported thatnumerous individual amino acid residues in both Ig-like domains of CD80are important for binding to CTLA-4 and CD28. Peach et al. (1995), J.Biol. Chem. 270(36): 21181-87. CD86 is constitutively expressed at lowlevels and is rapidly upregulated after activation, whereas CD80 isinducibly expressed later after activation. Sharpe and Freeman (2002),Nature Reviews Immunology 2: 116-26.

Another T cell regulatory molecule, referred to herein as B7RP-1, has aplethora of names, KIAA0653 (Ishikawa et al. (1998), DNA Res. 5:169-76), B7h (Swallow et al. (1999), Immunity 11: 423-32), GL50 (Ling etal. (2000), J. Immunol. 164: 1653-57), B7RP-1 (Yoshinaga et al. (1999),Nature 402: 827-32), LICOS (Brodie et al. (2000), Curr. Biol. 10:333-36), B7-H2 (Wang et al. (2000), Blood 96:2808-13), and ICOSL (Sharpeand Freeman, supra). B7RP-1 is expressed in peripheral lymphoid tissues,spleen, lymph nodes, lung, thymus, splenocytes, and B cells. Interactionof B7RP-1 with T cells can increase T cell proliferation and cytokineproduction. B7RP-1 signals T cells through the ICOS receptor, which isexpressed on activated T cells. Yoshinaga et al. (1999), Nature 402:827-32; Swallow et al. (1999), Immunity 11: 423-32. B7RP-1 isconstitutively expressed in unstimulated B cell lines, and itsexpression can be induced in monocytes by interferon γ. Aicher et al.(2000), J. Immunol. 164: 4689-96. The development and regulatoryfunction of regulatory T cells is dependent on the interaction betweenB7RP-1 and its receptor on T cells. Akbari et al. (2002), NatureMedicine 8(9):1024-32.

Further, three other T cell regulatory molecules, PD-L1 (also calledB7-H1), PD-L2 (also called B7-DC), and B7-H4 (also called B7S1 and B7x)can inhibit T cell proliferation and cytokine production. PD-L1 andPD-L2 act through their common receptor, PD-1, which is expressed on Tcells, B cells, and myeloid cells. Freeman et al. (2000), J. Exp. Med.192(7): 1027-34; Dong et al. (1999), Nature Medicine 5(12): 1365-69;Latchman et al. (2001), Nature Immunology 2(3): 261-68; Tamura et al.(2001), Blood 97(6): 1809-16; and Tseng et al. (2001), J. Exp. Med.193(7): 839-45. Expression of PD-L1 and PD-L2 can be induced byinterferon γ, a generally pro-inflammatory cytokine Latchman et al.,supra. B7-H4 is expressed on B cells, macrophages, and dendritic cellsand likely acts through BTLA, an inhibitory receptor expressed on B andT cells. Watanabe et al. (2003), Nature Immunology 4(7): 670-79; Zang etal. (2003), Proc. Natl. Acad. Sci. 100(18): 10388-92; Sica et al.(2003), Immunity 18: 849-61; Prasad et al. (2003), Immunity 18: 863-73;Carreno and Collins (2003), Trends Immunol. 24(10): 524-27.

Still another butyrophilin-like protein that plays a costimulatory rolein stimulating T cells is B7-H3. Chapoval et al. (2001), Nature Immunol.2(3):269-74. Like the other B7 family members, B7-H3 has a signalsequence, extracellular Ig-like domains, a transmembrane domain, and acytoplasmic domain. The human B7-H3 gene encodes isoforms with two orfour extracellular Ig-like domains. Sun et al. (2002), J. Immunol. 168:6294-97. Expression of B7-H3 can be induced on dendritic cells byinflammatory cytokines B7-H3 can stimulate proliferation and thecytotoxic response of T cells. B7-H3 acts through a putative T cellreceptor that is distinct from CD28, CTLA-4, ICOS, and PD-1. Chapoval etal, supra.

BTL-II Proteins

The existence of human and murine BTL-II proteins has been predictedfrom genomic sequence. Stammers et al. (2000), Immunogenetics 51:373-82. However, these authors found no evidence of a transmembrane or acytoplasmic domain in human or murine BTL-II proteins based on genomeanalysis and no evidence of a transcript connecting exons 1-4 (whichencode a signal peptide, two Ig-like domains, and a heptad repeatregion, respectively) with exons 5 and 6 (which encode another twoIg-like domains, respectively) in PCR experiments designed to detectmurine BTL-II mRNAs. Stammers et al., supra. Based on these findings,BTL-II was not placed in the B7 subfamily of cell surface,immunomodulatory proteins. Later sequence submissions to publicdatabases by these same authors predict a human BTL-II mRNA of 1368nucleotides, which includes exons 5 and 6, encoding a protein of 455amino acids, which lacks a transmembrane domain and a cytoplasmic domain(NCBI accession no. NM_(—)019602, which discloses SEQ ID NO:1 (humanBTL-II cDNA sequence) and SEQ ID NO:2 (human BTL-II protein sequence)).

The BTL-II nucleic acid and protein sequences of the invention differfrom these sequences in a number of respects. The cDNA sequence encodesa protein comprising a signal sequence, an extracellular domain, atransmembrane domain, and a cytoplasmic domain, unlike the previouslyreported BTL-II protein sequence, which contained no transmembrane orcytoplasmic domains. These characteristics, along with the expressionpattern of BTL-II, place BTL-II within the B7 subfamily ofbutyrophilin-like proteins. Table 1 (below) highlights the differencesbetween the BTL-II protein of the invention and the previously reportedsequence. A BTL-II protein of the invention is shown on the top line(SEQ ID NO:4), and the BTL-II protein reported in NCBI accession no.NM_(—)019602 is shown on the bottom line (SEQ ID NO:2). From thiscomparison, it is apparent that there are three mismatches between thetwo sequences (at positions 360, 454, and 455) and that SEQ ID NO:4 has27 more amino acids, which constitute additional sequence in theextracellular domain as well as a transmembrane and a cytoplasmicdomain. These sequences 99.3% identical according to the GAP programusing the parameters recited above.

TABLE 1 Comparison of human BTL-II predicted protein sequences

Besides an overall similarity in domain structure as illustrated in FIG.1, the B7 subfamily proteins have similarities at the primary sequencelevel. For example, when aligned pairwise using the computer programGAP, the human BTL-II protein sequence (SEQ ID NO:4) is similar to otherB7 subfamily members as displayed in Table 2 below.

TABLE 2 Percent identity to Percent similarity human BTL-II to humanBTL-II protein protein Human PD-L1 (NCBI 19% 28% accession no.NP_054862) Human PD-L2 (NCBI 19% 29% accession no. NP_079515) Human CD80(NCBI 26% 33% accession no. P33681) Human CD86 (NCBI 23% 32% accessionno. P42081) Murine BTL-II 62% 68%One of skill in the art will realize that residues that are conserved inany of these alignments are more likely to play an essential role in thestructure or function of human BTL-II than those that are not conserved.

In Table 3 (below), the human BTL-II amino acid sequence (SEQ ID NO:4,top line) is aligned with the murine BTL-II amino acid sequence (SEQ IDNO:6; bottom line). Identical amino acids are joined by a vertical line,and similar amino acids have one or two dots between them. The percentidentity between these sequences as determined GAP (described above) isabout 62%, and the percent similarity is about 68%. Residues found inIgV- or IgC-like domains or in the so-called “I set” of IgV-likeimmunoglobulin superfamily members or conservative substitutions of suchresidues are shown in boldface. Peach et al., supra; Harpaz and Chothia(1994), J. Mol. Biol. 238: 528-39. Such residues are likely to bestructurally important and, thus, may have functional effects. Theoccurrence of a substantial number of such amino acids in the properspacing can identify a sequence as IgV-like or IgC-like.

TABLE 3

One of skill in the art will appreciate that non-conserved residues areless likely to play a role in determining the overall tertiary structureof a BTL-II protein than conserved residues, since structure is moreconserved in evolution than sequence. Bork et al. (1994), J. Mol. Biol.242: 309-20. As used herein, “non-conserved residues” are amino acidswithin a BTL-II protein that are not conserved when the human and themurine BTL-II protein sequences are compared as in Table 3. In BTL-IIproteins encoded by splice variants, such residues will occur adifferent numerical positions within the sequence. For example, thenon-conserved residue at position 397 of SEQ ID NO:4 is the samenon-conserved residue seen at position 187 of SEQ ID NO:8. One of skillin the art will appreciate that protein structure can affect proteinfunction. Further, non-conserved amino acids are also less likely toplay a direct role in BTL-II function. For example, residues 4, 6, 25,26, 35, 36, and many others are neither identical nor similar. Thus, oneof skill in the art would realize that alteration of such residues wouldbe less likely to affect BTL-II protein function that would alterationof conserved or similar residues. Moreover, conservative substitutionsare less likely to affect protein function that non-conservativesubstitutions. Examples of amino acid substitutions that areconservative substitutions, unlikely to affect biological activity,including the following: Ala for Ser, Val for Ile, Asp for Glu, Thr forSer, Ala for Gly, Ala for Thr, Ser for Asn, Ala for Val, Ser for Gly,Tyr for Phe, Ala for Pro, Lys for Arg, Asp for Asn, Leu for Ile, Leu forVal, Ala for Glu, Asp for Gly, and these changes in the reverse. Seee.g. Neurath et al., The Proteins, Academic Press, New York (1979).Further, an exchange of one amino acid within a group for another aminoacid within the same group is a conservative substitution where thegroups are the following: (1) alanine, valine, leucine, isoleucine,methionine, norleucine, and phenylalanine; (2) histidine, arginine,lysine, glutamine, and asparagine; (3) aspartate and glutamate; (4)serine, threonine, alanine, tyrosine, phenylalanine, tryptophan, andcysteine; (5) glycine, proline, and alanine;

Human BTL-II protein comprises several recognizable domains. First, is asignal sequence (encoded by exon 1), which extends from residue 1 of SEQID NO:4 to a second position from about residue 22 to 29 of SEQ ID NO:4.Next is an Ig-like domain (encoded by exon 2), which extends fromresidue x to residue y, where x is from residue 22 to 32 and y is fromresidue 138 to 148 of SEQ ID NO:4. Following this is another Ig-likedomain (encoded by exon 3) extending from residue v to residue w, whereresidue v is from residue 138 to 148 and w is from residue 232 to 242 ofSEQ ID NO:4. Next is a heptad repeat region (encoded by exon 4) fromresidue t to residue u, where t is from 234 to 239 and u is from residue240 to 247 of SEQ ID NO:4. Another Ig-like region extends from residue rto residue s, where r is from residue 240 to 247 and s is from residue354 to 364 of SEQ ID NO:4. A fourth Ig-like region extends from residuep to residue q, where p is from residue 355 to 365 and q is from residue452 to 462 of SEQ ID NO:4. These first six domains of human BTL-II makeup the extracellular region of human BTL-II. The signal sequence may,but need not, be cleaved from the rest of the protein upon secretion ofthe protein. A transmembrane domain extends from residue n to residue o,where n is from residue 454 to 462 and o is from residue 473 to 481 ofSEQ ID NO:4. Finally, a cytoplasmic domain extends from residue k toresidue m, where k is from residue 474 to 481 and m is at about residue482 of SEQ ID NO:4.

Murine BTL-II protein comprises a similar set of domains. First is asignal sequence starting at residue 1 and ending at a position fromabout residue 20 to about residue 27 of SEQ ID NO:6. Second is anIg-like domain extending from residue x to y, where x is from residue 21to 27 and y is from residue 138 to 148 of SEQ ID NO:6. Third is anotherIg-like domain from residue v to w, where v is from 139 to 148 and w isfrom 232 to 242 of SEQ ID NO:6. A heptad repeat region extends fromresidue t to u, where t is from 233 to 242 and u is from 238 to 248 ofSEQ ID NO:6. Another Ig-like region extends from residue r to s, where ris from 239 to 248 and s is from 355 to 365 of SEQ ID NO:6. A finalIg-like region extends from residue p to q, where p is from 356 to 365and q is from 447 to 457 of SEQ ID NO:6. These first six domains make upthe extracellular region of murine BTL-II. A transmembrane domainextends from residue n to o, where n is from 448 to 459 and o is from470 to 478 of SEQ ID NO:6. Finally, a cytoplasmic domain extends fromresidue k to m, where is from 471 to 478 and m is at about 514 of SEQ IDNO:6.

The instant invention encompasses secreted, soluble versions of BTL-IIas well as versions comprising a transmembrane domain that can beexpressed on a cell surface. The invention further includes BTL-IIproteins encoded by the BTL-II nucleic acids described below.Recombinant versions of all of these proteins can be used to produceantibodies, in screening, and/or as therapeutic agents as describedherein. For example, the invention encompasses BTL-II proteinscomprising all or part of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18.BTL-II proteins of the invention include proteins that differ from SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18 by insertion, deletion, alteration,or substitution in the primary amino acid sequence. Such variantsequences are at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or99.7% identical to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18, andcontain no internal gaps of over 10 amino acids when aligned using GAPwith the above-mentioned sequences. Examples of such sequences includethe naturally-occurring human allelic variants of BTL-II shown in FIGS.5 b, 6 b, and 7 b. If such variant sequences contain amino substitutionswhen compared to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18, thesesubstitutions can be conservative amino acid substitutions. Further,variant BTL-II proteins may contain no more than 3, 4, 5, 6, 7, 8, 9,10, 15, 20, or 25 insertions, deletions, or substitutions of a singleamino acid with respect to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ IDNO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18.

The BTL-II proteins of the invention include proteins encoded by varioussplice variants of the human and mouse BTL-II mRNA. Sequences of suchvariant human BTL-II proteins are shown in SEQ ID NO:8, SEQ ID NO:10,SEQ ID NO:12, SEQ ID NO:14, and SEQ ID NO:16. The sequence of a variantmouse BTL-II protein is shown in SEQ ID NO:18. Human splice variantslack either exon 3 alone or lack both exons 2 and 3, and the murinesplice variant disclosed lacks exon 3 only. See SEQ ID NO:7, SEQ IDNO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:17; FIGS.3 a, 4 a, 5 a, 6 a, 7 a, and 9 a. Proteins encoded by these sequencesand substantially similar sequences, where an alignment of the proteinsequence with at least one of the group consisting of SEQ ID NO:4, SEQID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, or SEQ ID NO:18 using GAP comprises no gaps longer than 10 aminoacids, are encompassed by the invention. If these proteins contain aminoacid substitutions relative to SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8,SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ IDNO:18, such substitutions are preferably conservative amino acidsubstitutions. BTL-II proteins of the invention can bind to a receptoron the surface of a T cell and can inhibit proliferation and/or cytokineproduction by T cells.

Further, the invention provides BTL-II proteins encoded by nucleic acidsthat span the splice junctions of exons 1 and 4 (SEQ ID NO:8 and SEQ IDNO:12) or exons 2 and 4 (SEQ ID NO:10, SEQ ID NO:14, SEQ ID NO:16, andSEQ ID NO:18) and substantially similar proteins that can bind to areceptor expressed on the surface of T cells and/or can inhibitproliferation and/or cytokine production by T cells. This specificallyincludes BTL-II proteins comprising a polypeptide consisting of an aminoacid sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.7%, or100% identical to amino acids 127 to 157 of SEQ ID NO:10, SEQ ID NO:14,or SEQ ID NO:18 or amino acids 126 to 156 of SEQ ID NO:16. The identityregion of the amino acid sequence aligned with amino acids 127 to 157 ofSEQ ID NO:10, SEQ ID NO:14, or SEQ ID NO:18 or amino acids 126 to 156 ofSEQ ID NO:16 is preferably at least 20, 23, 25, 27, 30, 35, or 40 aminoacids long. Such an amino acid sequence can be at least 150 amino acidslong and can be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.7%,or 100% identical to amino acids 30 to 358 of SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18. The identity region of the aminoacid sequence aligned with amino acids 30 to 358 of SEQ ID NO:10, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18 can be at least 50, 75, 100, 125,150, 175, 200, or 300 amino acids.

The invention also provides BTL-II proteins comprising a polypeptideconsisting of an amino acid sequence at least 80%, 85%, 90%, 95%, 96%,97%, 98%, 99%, 99.7%, or 100% identical to amino acids 30 to 457 of SEQID NO:4 that contains no more or less than 2 Ig-like domains and thatcan bind to a cell surface receptor expressed on B cells or T cells. Theamino acid sequence can be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%,99%, 99.7%, or 100% identical to amino acids 30 to 247 of SEQ ID NO:8 orto amino acids 30 to 243 of SEQ ID NO:12.

In further embodiments, the invention provides proteins encoded bynucleic acids comprising a polynucleotide that is 80%, 85%, 90%, 95%,96%, 97%, 98%, 99%, 99.7%, or 100% identical to a polynucleotideconsisting of nucleotides 34 to 124 of SEQ ID NO:11, where the identityregion is at least 60, 70, 80, 90, or 100 nucleotides long. MatureBTL-II proteins encoded by such polynucleotides may, but need not, lacka signal sequence (which is present in the immature version of theprotein) that is at least partially encoded by this polynucleotide. Suchproteins can bind to a T cell and/or can inhibit proliferation and/orcytokinine production by the T cell.

BTL-II proteins may be glycosylated to varying degrees or notglycosylated. As an illustration, a BTL-II protein of the invention maycomprise one or more N- or O-linked glycosylation sites in addition tothose already found in a protein comprising SEQ ID NO:4, SEQ ID NO:6,SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16,and/or SEQ ID NO:18. Such a BTL-II protein can have a longer in vivohalf life than an unaltered protein since it may have more sialic acidmoieties attached to it. BTL-II proteins also include proteinscomprising any one, any two, any three, or all four of the Ig-likedomains of a human or murine BTL-II or substantially similar domains.

Variants

Polypeptides derived from any BTL-II protein by any type of alteration(for example, but not limited to, insertions, deletions, orsubstitutions of amino acids; changes in the state of glycosylation ofthe polypeptide; refolding or isomerization to change itsthree-dimensional structure or self-association state; and changes toits association with other polypeptides or molecules) are also BTL-IIproteins as meant herein. The BTL-II proteins provided by the inventioninclude polypeptides characterized by amino acid sequences substantiallysimilar to those of the BTL-II proteins SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQID NO:18 that can bind to a receptor expressed on the surface of T cellsand/or can inhibit proliferation of and/or cytokine production of a Tcell. The region of identity can start at position 30 or higher of SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18. A GAP alignment of such a variantprotein with at least one of SEQ ID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, and/or SEQ ID NO:18may have no internal gaps longer than 10 amino acids. The portion of theBTL-II protein that is substantially similar to SEQ ID NO:4, SEQ IDNO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ IDNO:16, or SEQ ID NO:18 can be at least 100, at least 125, at least 150,at least 175, at least 200, or at least 250 amino acids long.Modifications in such proteins can be naturally provided or deliberatelyengineered. For example, SEQ ID NO:11 (FIG. 6 a) is an allelic orpolymorphic variant of SEQ ID NO:9 (FIG. 4 a) containing base changes ata handful of positions including at positions.

The invention provides BTL-II protein variants that contain single ormultiple amino acid alterations, which can be insertions, deletions, orsubstitutions of a single amino acid, relative to the sequence of SEQ IDNO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18, wherein the BTL-II variant proteincan inhibit T cell proliferation and/or cytokine production. Thealterations can be conservative amino acid substitutions. One of skillin the art is guided as to what amino acids can be changed withoutaffecting function by, for example, the alignment of human and mouseBTL-II proteins shown in Table 3. Amino acids that are identical orsimilar in human and mouse BTL-II are more likely to be important forfunction that those that are not. Further, amino acids that are highlyconserved in IgV or IgC domains (shown in boldface in Table 3) are alsolikely to be functionally important. Such allelic or polymorphicvariants can be valuable as diagnostic agents to determine apredisposition to disease. Human allelic variants with sequences varyingfrom SEQ ID NO:3 are disclosed in FIGS. 5 a, 5 b, 6 a, 6 b, 7 a, and 7b. For example, among normal people without symptoms of inflammatorybowel disease, a certain ratio can exist between the number havingBTL-II genes encoding cDNAs with the sequence of SEQ ID NO:3 and thenumber having BTL-II genes encoding the allelic variant mutationslabeled 3-6 in FIGS. 5 a, 6 a, and 7 a. The ratio of the occurrence ofthese alleles may be altered among patients with symptoms ofinflammatory bowel disease such that a greater proportion of thesepeople have BTL-II genes with the allelic variant mutations labeled 3-6in FIGS. 5 a, 6 a, and 7 a. The existence of various allelic variationsin a patient's tissues can be determined by, for example, PCRamplification of a segment of a DNA or RNA molecule spanning thepolymorphic site. As mentioned above, conservative amino acidsubstitutions, particularly at sites not conserved between human andmouse BTL-II protein sequences, are more likely to preserve biologicalfunction that are non-conservative substitutions at conserved sites. Oneof skill in the art will also appreciate that substitutions thatsubstantially upset the tertiary structure of a BTL-II protein aspredicted by programs such as, for example, DALI (Holm and Sander(1993), J. Mol. Biol. 233: 123-38), are likely to also impair function.

Fragments

Included among BTL-II proteins are proteins comprising fragments of SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18 or fragments that are at least 80%,85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% identical to thesesequences. Preferably, such fragments can bind to a receptor expressedon the surface of a T cell and are at least about 50, 60, 70, 80, 90,100, 150, or 200 amino acids long. Preferably, such fragments aresoluble in aqueous solution and comprise part or all of theextracellular region of a BTL-II protein. The protein comprising such afragment can be secreted. For example, BTL-II proteins comprising atleast one of the domains of the human or murine BTL-II proteinsdescribed above or a substantially similar protein, wherein the domainhas at least one of the biological properties of BTL-II proteins, areencompassed by the invention. For example, such proteins can inhibitproliferation and/or cytokine production of T cells.

Further, encompassed by the invention are the following altered versionsthe human and murine BTL-II proteins or substantially similar proteins:(1) versions of the human and murine BTL-II protein lacking the secondIg-like domain (see FIG. 1) encoded by exon 3; (2) versions of a humanor murine BTL-II protein lacking the first and second Ig-like domainsencoded by exons 2 and 3; (3) versions lacking third and fourth Ig-likedomains encoded by exons 5 and 6; (4) versions lacking any two of thefour Ig-like domains; and (5) versions lacking any one of the Ig-likedomains.

Also encompassed within the invention are immunogenic fragments of SEQID NO:4, SEQ ID NO:6, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ IDNO:14, SEQ ID NO:16, or SEQ ID NO:18 that are capable of elicitingantibodies that bind specifically to the fragment. Such fragments arepreferably at least 10 amino acids long and preferably comprisecontiguous amino acid residues from sequences mentioned above.Antibodies generated by immunizing animals with such fragments can beuseful for prediction, diagnosis, and treatment of inflammatory boweldiseases, as discussed elsewhere in this application. Such fragments canspan regions of these proteins encoded by splice junctions, which mayhave the advantage of generating antibodies specific for proteinsencoded by splice variants or the full length protein. Alternatively, anantibody against a portion of BTL-II that is encoded by exon 3 woulddetect only full length BTL-II proteins, not proteins encoded by anyother splice variants, all of which lack exon 3.

Recombinant Fusion Proteins

The invention further encompasses fusion proteins comprising at leastone BTL-II polypeptide, which is one of the BTL-II proteins, variants,or fragments described above, and at least one other moiety. The othermoiety can be a heterologous polypeptide, that is, a polypeptide otherthan a BTL-II polypeptide. The other moiety can also be a non-proteinmoiety such as, for example, a polyethylene glycol (PEG) moiety or acytotoxic, cytostatic, luminescent, and/or radioactive moiety.Attachment of PEG has been shown to increase the in vivo half life of atleast some proteins. Moreover, cytotoxic, cytostatic, luminescent,and/or radioactive moieties have been fused to antibodies for diagnosticor therapeutic purposes, for example, to locate, to inhibitproliferation of, or to kill cells to which the antibodies can bind.Similarly, BTL-II polypeptides fused to such moieties can be used tolocate, to inhibit proliferation of, or to kill cells that BTL-II canbind to. Among such cytotoxic, cytostatic, luminescent, and/orradioactive moieties are, for example, maytansine derivatives (such asDM1), enterotoxins (such as a Staphlyococcal enterotoxin), iodineisotopes (such as iodine-125), technetium isotopes (such as Tc-99m),cyanine fluorochromes (such as Cy5.5.18), ribosome-inactivating proteins(such as bouganin, gelonin, or saporin-S6), and calicheamicin, acytotoxic substance that is part of a product marketed under thetrademark MYLOTARG™ (Wyeth-Ayerst).

A variety of heterologous polypeptides can be fused to a BTL-IIpolypeptide for a variety of purposes such as, for example, to increasein vivo half life of the protein, to facilitate identification,isolation and/or purification of the protein, to increase the activityof the protein, and to promote oligomerization of the protein. Sincesome proteins of the B7 subfamily, such as, for example, CD80, bind totheir receptors primarily in oligomeric form (dimeric in the case ofCD80; see Collins et al. (2002), Immunity 17: 201-10), oligomerizationcan be very important to preserve the biological activity of a solubleprotein.

Many heterologous polypeptides can facilitate identification and/orpurification of recombinant fusion proteins of which they are a part.Examples include polyarginine, polyhistidine, or HAT™ (Clontech), whichis a naturally-occurring sequence of non-adjacent histidine residuesthat possess a high affinity for immobilized metal ions. Proteinscomprising these heterologous polypeptides can be purified by, forexample, affinity chromatography using immobilized nickel or TALON™resin (Clontech), which comprises immobilized cobalt ions. See e.g. Knolet al. (1996), J. Biol. Chem. 27(26): 15358-15366. Heterologouspolypeptides comprising polyarginine allow effective purification by ionexchange chromatography. Other useful heterologous polypeptides include,for example, the antigenic identification peptides described in U.S.Pat. No. 5,011,912 and in Hopp et al. (1988), Bio/Technology 6:1204. Onesuch peptide is the FLAG® peptide, which is highly antigenic andprovides an epitope reversibly bound by a specific monoclonal antibody,enabling rapid assay and facile purification of expressed recombinantfusion protein. A murine hybridoma designated 4E11 produces a monoclonalantibody that binds the FLAG® peptide in the presence of certaindivalent metal cations, as described in U.S. Pat. No. 5,011,912. The4E11 hybridoma cell line has been deposited with the American TypeCulture Collection under accession no. HB 9259. Monoclonal antibodiesthat bind the FLAG® peptide can be used as affinity reagents to recovera polypeptide purification reagent that comprises the FLAG® peptide.Other suitable protein tags and affinity reagents are: 1) thosedescribed in GST-Bind™ system (Novagen), which utilizes the affinity ofglutathione-S-transferase fusion proteins for immobilized glutathione;2) those described in the T7-Tag® affinity purification kit (Novagen),which utilizes the affinity of the amino terminal 11 amino acids of theT7 gene 10 protein for a monoclonal antibody; or 3) those described inthe Strep-tag® system (Novagen), which utilizes the affinity of anengineered form of streptavidin for a protein tag. Some of theabove-mentioned protein tags, as well as others, are described inSassenfeld (1990), TIBTECH 8: 88-93, Brewer et al., in Purification andAnalysis of Recombinant Proteins, pp. 239-266, Seetharam and Sharma(eds.), Marcel Dekker, Inc. (1991), and Brewer and Sassenfeld, inProtein Purification Applications, pp. 91-111, Harris and Angal (eds.),Press, Inc., Oxford England (1990). Further, fusions of two or more ofthe tags described above, such as, for example, a fusion of a FLAG tagand a polyhistidine tag, can be fused to a BTL-II protein of theinvention.

Recombinant fusion proteins comprising other heterologous polypeptidesmay have other kinds of unique advantages, such as, for example, apropensity to form dimers, trimers, or higher order multimers, anincreased in vivo half-life, and/or an increased biological activity.Techniques for preparing fusion proteins are known, and are described,for example, in WO 99/31241 and in Cosman et al. ((2001). Immunity 14:123-133). As an illustration, a heterologous polypeptide that comprisesan Fc region of an IgG antibody, or a substantially similar protein, canbe fused to a BTL-II polypeptide or fragment. An Fc region of anantibody is a polypeptide comprising C_(H)2 and C_(H)3 domains from anantibody of human or animal origin or immunoglobulin domainssubstantially similar to these. For discussion, see Hasemann and Capra,Immunoglobulins: Structure and Function, in William E. Paul, ed.,Fundamental Immunology, Second Edition, 212-213 (1989). Truncated formsof Fc regions comprising the hinge region that promotes dimerization canalso be used. Other portions of antibodies and other immunoglobulinisotypes can be used. Recombinant fusion proteins comprising Fc regionsof IgG antibodies are likely to form dimers. Fusion proteins comprisingvarious portions of antibody-derived proteins have been described byAshkenazi et al. ((1991) Proc. Natl. Acad. Sci. USA 88:10535-39), Byrnet al. ((1990), Nature 344: 677-70), Hollenbaugh and Aruffo (in CurrentProtocols in Immunology, Suppl. 4, pp. 10.19.1-10.19.11 (1992)), Baum etal. ((1994), EMBO J. 13: 3992-4001) and in U.S. Pat. No. 5,457,035 andWO 93/10151. In some embodiments, an altered Fc region can have theadvantage of having a lower affinity for Fc receptors compared to a wildtype Fc region. This is an advantage because it may lessen the lysis ofcells to which such recombinant fusion proteins bind by immune effectorcells. Example 5 describes the production of a fusion protein containingthe extracellular region of murine BTL-II fused to a human Fc region.The nucleic acid sequence encoding this protein and its amino acidsequence are disclosed in SEQ ID NO:19 and SEQ ID NO:20, respectively.

As another alternative, recombinant fusion proteins of the invention cancomprise a heterologous polypeptide comprising a leucine zipper. Amongknown leucine zipper sequences are sequences that promote dimerizationand sequences that promote trimerization. See e.g. Landschulz et al.(1988), Science 240: 1759-64. Leucine zippers comprise a repetitiveheptad repeat, often with four or five leucine residues interspersedwith other amino acids. Use and preparation of leucine zippers arewell-known in the art.

Alternatively, a heterologous polypeptide forming part of a recombinantfusion protein can be one or more peptide linkers, connecting two ormore BTL-II polypeptides. Generally, a peptide linker is a stretch ofamino acids that serves to link plural identical, similar, or differentpolypeptides to form multimers and provides the flexibility or rigidityrequired for the desired function of the linked portions of the protein.Typically, a peptide linker is between about 1 and 30 amino acids inlength. Examples of peptide linkers include, but are not limited to,—Gly-Gly—, GGGGS (SEQ ID NO:21), (GGGGS)n (SEQ ID NO:22), GKSSGSGSESKS(SEQ ID NO:23), GSTSGSGKSSEGKG (SEQ ID NO:24), GSTSGSGKSSEGSGSTKG (SEQID NO:25), GSTSGSGKSSEGKG (SEQ ID NO:26), GSTSGSGKPGSGEGSTKG (SEQ IDNO:27), or EGKSSGSGSESKEF (SEQ ID NO:28). Linking moieties aredescribed, for example, in Huston, J. S., et al., Proc. Natl. Acad. Sci.85: 5879-83 (1988), Whitlow, M., et al., Protein Engineering 6: 989-95(1993), and Newton, D. L., et al., Biochemistry 35: 545-53 (1996). Othersuitable peptide linkers are those described in U.S. Pat. Nos. 4,751,180and 4,935,233.

Further, a recombinant fusion protein can comprise a BTL-II protein thatlacks its normal signal sequence and has instead a heterologous signalsequence replacing it. The choice of a signal sequence depends on thetype of host cells in which the recombinant protein is to be produced,and a heterologous signal sequence can replace the native signalsequence. Examples of signal sequences that are functional in mammalianhost cells include the following: the signal sequence for interleukin-7(IL-7) described in U.S. Pat. No. 4,965,195; the signal sequence forinterleukin-2 receptor described in Cosman et al. ((1984), Nature 312:768); the interleukin-4 receptor signal peptide described in EP PatentNo. 0 367 566; the type I interleukin-1 receptor signal peptidedescribed in U.S. Pat. No. 4,968,607; and the type II interleukin-1receptor signal peptide described in EP Patent No. 0 460 846.

BTL-II Nucleic Acids

The invention encompasses isolated nucleic acids that encode the BTL-IIproteins, fragments, or immunogenic fragments described above, includingvariants, fragments, recombinant fusion proteins, full-length proteins,soluble proteins, and secreted proteins. These nucleic acids are usefulfor, inter alia, producing recombinant proteins and detecting thepresence of BTL-II nucleic acids in tissue samples, e.g. for diagnosticuses. Such nucleic acids can be genomic DNA or cDNA. The nucleic acidcan comprise an uninterrupted open reading frame encoding a BTL-IIprotein of the invention. Nucleic acid molecules of the inventioninclude DNA and RNA in both single-stranded and double-stranded form, aswell as the corresponding complementary sequences. An “isolated nucleicacid” is a nucleic acid that has been separated from adjacent geneticsequences present in the genome of the organism from which the nucleicacid was isolated, in the case of nucleic acids isolated fromnaturally-occurring sources. In the case of nucleic acids synthesizedchemically, such as oligonucleotides, or enzymatically from a template,such as polymerase chain reaction (PCR) products or cDNAs, it isunderstood that the nucleic acids resulting from such processes areisolated nucleic acids. An isolated nucleic acid molecule refers to anucleic acid molecule in the form of a separate fragment or as acomponent of a larger nucleic acid construct.

Further, the invention encompasses fragments of a nucleic acid encodinga BTL-II protein that can serve (1) as probes for detecting BTL-IInucleic acids by a number of methods well known in the art, e.g.Southern and northern blotting, dot blotting, colony hybridizations,etc., (2) as polymerase chain reaction (PCR) primers to amplify BTL-IInucleic acids, or (3) as a means to regulate expression of BTL-IInucleic acids, e.g. through inhibition of expression with antisensenucleic acids (including peptide nucleic acids), ribozymes, triplehelix-forming molecules, or interfering RNAs or DNAs that encode any ofthese RNAs. PCR primers can comprise, in addition to BTL-II nucleic acidsequences, other sequences such as restriction enzyme cleavage sitesthat facilitate the use of the amplified nucleic acid. PCR is describedin the following references: Saiki et al. (1988), Science 239:487-91;PCR Technology, Erlich, ed., Stockton Press, (1989). As explained below,PCR can be useful to detect overexpression of BTL-II mRNAs, and PCRprimers can be taken from various parts of the gene and can also beselected to distinguish between different splice variants. AntisenseRNAs (and DNAs encoding them), DNAs, or synthetic nucleotides and theiruse to regulate expression are well known in the art and are describedin, e.g. Izant and Weintraub (1984), Cell 36(4): 1007-15; Izant andWeintraub (1985), Science 229(4711): 345-52; Harel-Bellan et al. (1988),J. Exp. Med. 168(6): 2309-18; Sarin et al. (1988), Proc. Natl. Acad.Sci. USA 85(20):7448-51; Zon (1988), Pharm. Res. 5(9): 539-49;Harel-Bellan et al. (1988), J. Immunol. 140(7): 2431-35; Marcus-Sekuraet al. (1987), Nucleic Acids Res. 15(14):5749-63; Gambari (2001), Curr.Pharm. Des. 7(17): 1839-62; and Lemaitre et al. (1987), Proc. Natl.Acad. Sci. USA 84(3): 648-52. Similarly, interfering RNAs (and DNAsencoding them) and their use to inhibit expression of selected genes arewell known in the art and described in, e.g., Fjose et al. (2001),Biotechnol. Ann. Rev. 7: 31-57; Bosher and Labouesse (2000), Nature CellBiol. 2:E31-E36. Further, ribozymes or DNAzymes can be targeted tocleave specific RNAs and thus used to inhibit gene expression asdescribed in, e.g., Lewin and Hauswirth (2001), Trends Mol. Med. 7(5):221-28; Menke and Hobom (1997), Mol. Biotechnol. 8(1): 17-33; Norris etal. (2000), Adv. Exp. Med. Biol. 465: 293-301; Sioud (2001), Curr. Mol.Med. 1(5): 575-88; and Santiago and Khachigian (2001), J. Mol. Med.79(12): 695-706. Nucleic acids that can regulate BTL-II expression canfind use in in vivo or in vitro studies of BTL-II function or astherapeutics, optionally as gene therapy agents.

The present invention also includes nucleic acids that hybridize undermoderately stringent conditions, and more preferably highly stringentconditions, to nucleic acids encoding the BTL-II proteins describedherein. Such nucleic acids include SEQ ID NO:3, SEQ ID NO:5, SEQ IDNO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, or SEQ IDNO:17. Preferably such nucleic acids encode proteins that can bind to areceptor on the surface of T cells and/or can inhibit T cellproliferation and/or cytokine production. Hybridization techniques arewell known in the art and are described by Sambrook, J., E. F. Fritsch,and T. Maniatis (Molecular Cloning: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11,(1989)) and Current Protocols in Molecular Biology (F. M. Ausubel etal., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4 (1995)).Moderately stringent conditions include hybridization in about 50%formamide, 6×SSC at a temperature from about 42 to 55° C. and washing atabout 60° C. in 0.5×SSC, 0.1% SDS. Highly stringent conditions aredefined as hybridization conditions as above, but with washing atapproximately 68° C. in 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15 M NaCl,10 mM NaH₂PO₄, and 1.26 mM EDTA, pH 7.4) can be substituted for SSC(1×SSC is 0.15 M NaCl and 15 mM sodium citrate) in the hybridization andwash buffers; washes, preferably at least two, are performed for 15minutes after hybridization is complete.

It should be understood that the wash temperature and wash saltconcentration can be adjusted as necessary to achieve a desired degreeof stringency by applying the basic principles that govern hybridizationreactions and duplex stability, as known to those skilled in the art anddescribed further below (see e.g., Sambrook et al., supra). When nucleicacids of known sequence are hybridized, the hybrid length can bedetermined by aligning the sequences of the nucleic acids andidentifying the region or regions of optimal sequence complementarity.The hybridization temperature for hybrids anticipated to be less than 50base pairs in length should be 5 to 10° C. less than the meltingtemperature (Tm) of the hybrid, where Tm is determined according to thefollowing equations. For hybrids less than 18 base pairs in length, Tm(degrees C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids above 18base pairs in length, Tm (degrees C.)=81.5+16.6(log₁₀ [Na⁺])+0.41(%G+C)−(600/N), where N is the number of bases in the hybrid, and [Na⁺] isthe concentration of sodium ions in the hybridization buffer ([Na⁺] for1×SSC=0.165 M). Each such hybridizing nucleic acid has a length that isat least 15 nucleotides (or at least 18 nucleotides, or at least 20, orat least 25, or at least 30, or at least 40, or at least 50, or at least100. Sambrook et al., supra.

BTL-II nucleic acids include nucleic acids comprising the followingpolynucleotides: (1) all or or a fragment of SEQ ID NO:3, SEQ ID NO:5,SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ ID NO:15, orSEQ ID NO:17, wherein the fragment encodes a BTL-II protein that canbind to a receptor expressed on the surface of a T cell and/or inhibitproliferation and/or cytokine production of T cells; (2) sequences atleast 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7% identical toSEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, or SEQ ID NO:17 that are at least 100, 125, 150,175, 200, 225, 250, 300, 400, 500, 600, 800, 1000. 1200, 1400, or 1600nucleotides long and encode a BTL-II protein that can bind to a receptorexpressed on the surface of T cells and/or inhibit proliferation and/orcytokine production of T cells; (3) fragments of SEQ ID NO:3, SEQ IDNO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ ID NO:13, SEQ IDNO:15, or SEQ ID NO:17 or substantially similar sequences that areuseful for detecting or amplifying nucleic acids encoding the BTL-IIproteins of the invention or for regulating the expression of BTL-IImRNAs and/or proteins; (4) nucleic acids comprising a polynucleotidethat is at least 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 99.7%identical to a polynucleotide consisting of nucleotides 30 to 130 of SEQID NO:7 or SEQ ID NO:11 or to nucleotide 377-477 of SEQ ID NO:9, SEQ IDNO:13, SEQ ID NO:15, or SEQ ID NO:17, wherein the region of identity isat least 60, 70, 80, 90, or 100 nucleotides long and the protein encodedby the nucleic acid can inhibit proliferation and/or cytokine productionof T cells; and (5) nucleic acids that comprise at least 1, 2, 3, 4, 6,8, 10, 15, 20, 25, 30, 35, 40, 50, or 75 alteration(s) relative to SEQID NO:3, SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, SEQ ID NO:15, or SEQ ID NO:17, wherein an alteration can be aninsertion, deletion or substitution of a single nucleotide.

Antibodies that Bind Specifically to BTL-II Polypeptides

Antibodies that bind specifically to the BTL-II proteins of theinvention, including variants, fragments, and recombinant fusionproteins, are encompassed by the invention. As used herein, specificbinding of an epitope on a BTL-II protein by another protein (such as anantibody) means that the specifically-bound protein can be displacedfrom the molecule of BTL-II protein to which it is bound by anotherprotein comprising the same epitope but not by another protein whichdoes not comprise this epitope. Numerous competitive binding assays areknown in the art. Epitopes may comprise only contiguous amino acids, butalso may comprise non-contiguous amino acids that are brought intoproximity by the tertiary folding of a BTL-II protein. Epitopes can beidentified by methods known in the art. See e.g. Leinonen et al. (2002),Clin. Chem. 48(12): 2208-16; Kroger et al. (2002), Biosens. Bioelectron.17(11-12): 937-44; Zhu et al. (2001), Biochem. Biophys. Res. Commun.282(4):921-27. The invention also encompasses epitopes of the BTL-IIproteins described herein that are useful for generating antibodies,which are referred to herein as immunogenic fragments. Immunogenicfragments are preferably at least 10 amino acids long and preferablycomprise contiguous amino acids from SEQ ID NO:4, SEQ ID NO:6, SEQ IDNO:8, SEQ ID NO:10, SEQ ID NO:12, SEQ ID NO:14, SEQ ID NO:16, or SEQ IDNO:18. Such epitopes can span regions of BTL-II proteins encoded bysplice junctions, which may have the advantage of specific binding toproteins encoded by specific splice variants.

Antibodies can be polyclonal or monoclonal antibodies and can beproduced by methods well-known in the art. See, for example, MonoclonalAntibodies, Hybridomas: A New Dimension in Biological Analyses, Kennetet al. (eds.), Plenum Press, New York (1980); and Antibodies: ALaboratory Manual, Harlow and Land (eds.), Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., (1988); Kohler and Milstein (1980)Proc. Natl. Acad. Sci., USA, 77: 2197; Kozbor et al. (1984), J. Immunol.133: 3001-3005 (describing the human B-cell hybridoma technique); Coleet al., Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc.,pp. 77-96 (1985)(which describes EBV-hybridoma technique);. Kuby,Immunology, Second Edition, p. 162-64, W.H. Freeman and Co., New York(1994). Hybridoma cell lines that produce monoclonal antibodies specificfor the BTL-II proteins of the invention are also contemplated herein.Such hybridomas can be produced and identified by conventionaltechniques. The hybridoma producing the mAb of this invention can becultivated in vitro or in vivo. Further, anti-BTL-II antibodies of theinvention can be produced in other cultured cells, including, forexample, Chinese hamster ovary (CHO), HeLa, VERO, BHK, Cos, MDCK, 293,3T3, myeloma (e.g. NSO, NSI), or WI38 cells, yeast cells, insect cells,and bacterial cells, including, for example, Eschericha coli. Suchantibodies can be produced by introducing nucleic acids encoding theantibodies plus nucleic acids to enable expression of these nucleicacids into desired host cells. The antibodies can then be produced byculturing the cells into which these nucleic acids have been introduced.Monoclonal antibodies can be of any immunoglobulin class including IgG,IgM, IgE, IgA, IgD and any subclass thereof.

Alternatively, antibodies can be single chain antibodies comprising aheavy and a light chain variable region-like domain and, optionally,also one or more constant region-like domain (U.S. Pat. No. 4,946,778;Bird et al. (1988), Science 242: 423-26; Huston et al. (1988), Proc.Natl. Acad. Sci. USA 85: 5879-83), dimeric or multivalent antibodies(see e.g. Lantto et al. (2002), J. Gen. Virol. 83: 2001-05; Hudson andSouriau (2001), Expert Opin. Biol. Ther. 1(5): 845-55), tetramericantibodies (see e.g. Janeway et al., Immunobiology: The Immune System inHealth and Disease, Fifth Edition, Part II, Ch. 3, Garland Publishing(2001)), chimeric antibodies (Hudson and Souriau, supra; Boulianne etal. (1984), Nature 312:643-46; Morrison et al (1984), Proc. Natl. Acad.Sci. USA 81: 6851-55; Takeda et al. (1985), Nature 314: 452-54;Neuberger et al. (1985), Nature 314: 268-70), fully human antibodiesproduced in a different transgenic mammal (described in e.g., U.S. Pat.No. 6,150,584) or by in vitro selection (US Patent Application No.2002/0058033) or humanized antibodies (Morrison et al., supra; Takeda etal., supra; Boulianne et al., supra). Further, antibodies can be“matured” by in vitro selection schemes to yield an antibody withaltered properties such as, for example, a higher affinity for theepitope to which it binds. See e.g. Jackson et al. (1995), J. Immunol.154(7): 3310-19; Pini and Bracci (2000), Curr. Protein Pept. Sci. 1(2):155-69; Ellmark et al. (2002), Mol. Immunol. 39(5-6): 349; O'Connell etal. (2002), J. Mol. Biol. 321(1): 49-56; Huls et al. (2001), CancerImmunol. Immunother. 50: 163-71; Hudson and Souriau, supra; Adams andSchier (1999), J. Immunol. Methods 231(1-2): 249-60; Schmitz et al.(2000), Placenta 21 Suppl. A: S106-12. Alternatively, fragments of anantibodies such as, for example, Fab fragments, F(ab′)₂ fragments, orsingle chain Fv fragments (scFv's) that can bind specifically to aBTL-II protein of the invention are also encompassed by what is meantherein as an anti-BTL-II antibody. See Kuby, supra, pp. 109-112 andJaneway et al., supra, for discussion of Fab and Fv fragments. Theinvention also encompasses anti-idiotypic antibodies that bindspecifically to antibodies that bind specifically to BTL-II proteins andthat mimic the effects of BTL-II proteins. Such anti-idiotypicantibodies find the same uses as BTL-II proteins. Methods for generatinganti-idiotypic antibodies are well known in the art. See e.g. Kuby etal., supra, at 371-72. Various kinds of recombinant and non-recombinantbispecific antibodies that can bind specifically to a BTL-II protein ofthe invention and another epitope are also contemplated. Various kindsof bispecific antibodies and methods for making them are described ine.g. U.S. Pat. Nos. 4,474,893, 6,060,285, and 6,106,833.

The anti-BTL-II antibodies may be antagonistic antibodies that block abiological function of BTL-II, such as the binding of BTL-II to itsreceptor, or agonistic antibodies that promote a biological function ofBTL-II or mimic the function of BTL-II. Agonistic antibodies can includeagonistic anti-idiotypic antibodies that mimic the function of BTL-IIprotein. Assays for BTL-II function are described herein. Anti-BTL-IIantibodies that block a biological function of BTL-II as determined insuch assays are antagonistic antibodies as meant herein. Antagonisticantibodies may, for example, block the binding of BTL-II to itsreceptor. Anti-BTL-II antibodies that, when added to such assays,promote or enhance a biological function of BTL-II are agonisticantibodies as meant herein. An antagonistic anti-BTL-II antibody can beused, for example, as an adjuvant to enhance a mucosal immune response.Further, an agonistic antibody against a BTL-II receptor can be used todepress a mucosal immune response to treat, for example, a disease thatis characterized by inappropriate inflammation of the gut, such asCrohn's disease or inflammatory bowel disease.

The antibodies of the invention can also be used in assays to detect thepresence of the BTL-II proteins of the invention, either in vitro or invivo. The antibodies also can be employed in purifying BTL-II proteinsof the invention by immunoaffinity chromatography.

The invention encompasses nucleic acids encoding the antibodies of theinvention and methods for producing the antibodies by introducing suchnucleic acids into cells and culturing the cells containing the nucleicacids.

Agonists and Antagonists of BTL-II Polypeptides

The invention comprises agonists and antagonists of BTL-II and methodsfor screening for and using agonists and antagonists. Assays for BTL-IIbiological activity are described herein such as, for example, cellproliferation assays, cytokine secretion assays, binding assays, andgenetic assays involving the over- or under-expression of BTL-II proteinin vivo or in vitro or a complete absence of BTL-II expression in vivoor in vitro. Candidate molecules can be added to such assays todetermine their effects on the biological activity of BTL-II proteins.BTL-II antagonists can, for example, block the interaction of BTL-IIwith its receptor, which is preferably expressed on B cell or T cells.Antagonists include antagonistic antibodies, and agonists includeagonistic antibodies. In addition, other antibody-related molecules thatcan bind specifically to the BTL-II proteins of the invention, such asaffibodies (Ronnmark et al. (2002), J. Immunol. Methods 261(1-2):199-211) and the biologically active peptides described in WO 00/24782that can bind specifically to the BTL-II proteins of the invention andinhibit the biological activity of BTL-II proteins are encompassed bythe invention. Further, BTL-II antagonists include the nucleic acidsdescribed above that are useful for modulating expression of BTL-IIprotein and/or mRNA, such as, for example, interfering RNAs (or DNAsthat encode them) or antisense RNAs or DNAs.

Antagonists further include proteins that comprise amino acid sequencesselected in vitro to bind to BTL-II or its receptor and that can,optionally, interfere with the interaction of BTL-II and its receptor.Alternatively, such proteins can be BTL-II agonists that promote ormimic the biological function of BTL-II. Proteins that bind to BTL-II orits receptor can be screened for their ability to interfere with theinteraction of BTL-II with its receptor, or, alternatively, a selectioncan be designed to obtain such proteins directly.

Proteins may be selected by a number of methods such as, for example,phage display or display of the surface of a bacterium. See e.g. Parmleyand Smith (1989), Adv. Exp. Med. Biol. 251: 215-218; Luzzago et al.(1995), Biotechnol. Annu Rev. 1:149-83; Lu et al. (1995), Biotechnology(NY) 13(4): 366-372. In these methods, each member of a library ofbinding domains can be displayed on individual phage particles orbacterial cells, and bacteria or phage that bind to a protein ofinterest under chosen conditions can be selected. Nucleic acids encodingthe selected binding domains can be obtained by growing the selectedphage or bacteria and isolating nucleic acids from them.

Alternatively, a protein can be selected entirely in vitro. For example,each individual polypeptide in a library of potential binding domainscan be attached to nucleic acids encoding it, and those that bind to theprotein of interest under chosen conditions can be selected. Since thepolypeptides are attached to nucleic acids encoding them, subsequentoperations, such as amplifying, cloning, or sequencing nucleic acidsencoding effective binding domains are facilitated. Various schemes forsuch selections are known in the art, including antibody-ribosome-mRNAparticles, ribosome display, covalent RNA-peptide fusions, or covalentDNA-RNA-peptide fusions. He and Taussig (1997), Nucleic Acids. Res.25(24): 5132-5134; Hanes and Pluckthun (1997), Proc. Natl. Acad. Sci.94: 4937-4942; Roberts and Szostak (1997), Proc. Natl. Acad. Sci. 94:12297-12302; Lohse and Wright (2001), Curr. Opin. Drug Discov. Devel.4(2): 198-204; Kurz et al. (2000), Nucleic Acids Res. 28(18): E83; Liuet al. (2000), Methods Enzymol. 318: 268-93; Nemoto et al. (1997), FEBSLett. 414(2): 405-08; U.S. Pat. No. 6,261,804; WO0032823; and WO0034784.Such proteins can be selected to be antagonists or agonists.

Assays for the Biological Activity of BTL-II Proteins

Various assays can be used to detect the biological activity of a BTL-IIprotein and to identify binding partners of BTL-II. BTL-II proteins,receptor(s) of BTL-II, and agonists and/or antagonists of either can beused in such assays.

Assays to Identify Binding Partners

A binding partner for BTL-II protein can be identified by firstdetermining what type of cells a soluble BTL-II protein can bind to.Since the known receptors for members of the B7 subfamily ofbutyrophilin-like proteins are all expressed on T cells, eitherconstitutively (CD28) or after activation (CTLA-4, ICOS, PD-1), T cellscan be tested to determine whether BTL-II can bind to them. Because ofthe expression pattern of BTL-II, T cells isolated from normal andinflamed gut can be included in such tests. In addition, various subsetsof T cells, including memory T cells, naïve T cells, αβ cells, γδ Tcells, and T cells in various activation states, can be tested.

Such experiments can be conducted using methods well known in the art.For example, a BTL-II recombinant fusion protein comprising theextracellular domain of murine BTL-II plus an Fc region of an antibodycan be used to bind to the cells being tested. A fluorescently-labeledantibody that can bind to the Fc region in the recombinant fusionprotein can be added. After washing, the cells can be analyzed using afluorescence activated cell sorting (FACS) device to determine whetherBTL-II can bind to the cells. One such assay is described by Chapoval etal.)(2001), Nature Immunol. 2(3): 269-74). Other methods known in theart can also be suitable to determine whether BTL-II binds to specificcell populations.

Further, known receptors of B7 subfamily members will be tested todetermine whether BTL-II binds to any of these proteins. Recombinantfusion proteins comprising an extracellular domain of a receptor of a B7sub-family member (such as, for example, CTLA-4, a receptor for B7-1 andB7-2) and an Fc region of an antibody can be made by methods similar tothose used to make such BTL-II fusion proteins. Cells can be transfectedwith full length forms of BTL-II nucleic acids encoding BTL-II proteinsincluding transmembrane and cytoplasmic domains that are expressed onthe cell surface. The receptor:Fc fusion protein to be tested can beadded to such cells along with a fluorescently-labeled antibody that canbind to the Fc region. After washing, the cells can be analyzed by FACSto determine whether the receptor:Fc fusion protein binds to the BTL-IIprotein expressed on the transfected cells. The reverse experiment canalso done where soluble BTL-II:Fc fusion protein is used and the fulllength receptor protein is introduced and expressed via transfection.Such experiments can reveal binding interactions between BTL-II proteinsand known receptors of other B7 subfamily members.

If BTL-II binds to at least one variety of T cells but does not bind toa known receptor, a variety of expression cloning or proteinpurification methods known in the art can be used to identify thereceptor that BTL-II binds to. As an example, a radioactive slidebinding cDNA expression cloning method can be used. Briefly, a cellsource with the greatest binding to soluble BTL-II is identified, mRNAis isolated, and a cDNA library is built in a mammalian expressionvector. Mammalian cells are transfected with pools of cDNAs on slides,and after an appropriate incubation to allow expression, solubleBTL-II:Fc fusion protein is bound to the cells. Specific binding toreceptor bearing cells is detected by the following series of steps:binding of a radioactive anti-Fc reagent to the bound BTL-II:Fc protein;application of film emulsion to the slides; incubation to allowexposure; film development to deposit silver grains; and detection ofthe grains by microscope. The receptor-expressing clone is then isolatedfrom the pool by sub-dividing the pool and iterative slide bindingassays to identify the single receptor clone. Such methods are describedin McMahon et al. (1991), EMBO J. 10: 2821-32.

When binding to cells is achieved, a variety of means, either throughanimal immunization or phage display technology, can be used to isolateantibodies that bind BTL-II and disrupt its binding to cells. Suchantibodies can be used to antagonize the activity of endogenous BTL-IIand therefore can be used in assays to determine the effects of loweredeffective amounts of BTL-II on immune responses and in disease models,such as the inflammatory bowel disease models, for example, thosedescribed by Cooper et al. ((1993), Lab. Invest. 69(2): 238-49) andTokoi et al. ((1996), J. Gastroenterol. 31(2): 182-88).

Assays to Determine BTL-II Function

B7 subfamily proteins have been shown to have activity in T cell“co-stimulatory” assays that involve activating T cells through their Tcell receptors with varying doses of “antigen.” The activity of the B7subfamily proteins is most evident at “sub-optimal” T cell receptorstimulation. See e.g. Latchman et al. (2001), Nature Immunol. 2(3):261-68. A “surrogate antigen,” such as tissue culture dish boundanti-CD3ε antibody or antigens presented by the MHC molecules onirradiated antigen presenting cells, can be used. A soluble BTL-IIprotein can be added to determine its effect on the T cells' response tothe “antigen.” Various parameters can be measured to determine whetherthe T cells are being stimulated or suppressed. Cellular proliferation,cell surface receptor expression, and levels of expression ofimmunomodulatory molecules (such as, for example, interferon γ and/orIL-2) at the protein and/or mRNA level can be measured. Such methods areused and described in e.g., Fitch et al. in T Cell Subsets in Infectiousand Autoimmune Diseases, John Wiley and Sons, pp. 68-85 (1995); Freemanet al. (2000), J. Exp. Med. 192(7):1027-34; Swallow et al. (1999),Immunity 11:423-32; Hutloff et al. (1999), Nature 397: 263-66; Yoshinagaet al. (1999), Nature 402: 827-32; Latchman et al., supra. Given theexpression pattern of BTL-II, such assays can include T cells derivedfrom mucosal tissues or the lymph nodes that drain such tissues. Inaddition, naïve and memory T cells, CD4⁺ and CD8⁺ T cells, and T cellsexpressing T cell receptors other than αβ T cell receptors can beexamined. From such experiments can reveal whether BTL-II can alterresponses of a number of distinct kinds of T cells. Such experiments aredescribed below and show that a soluble version of BTL-II can inhibitcell proliferation and suppress production of cytokines, includinginterferon gamma (IFNγ), interleukin 2 (IL2), and interleukin 5 (IL5).Examples 6-10; FIGS. 12-17.

Variations of this kind of experiment include examining the effect ofincluding soluble forms of BTL-II proteins on the co-stimulatoryresponse found with other soluble B7 subfamily proteins or various TNFfamily members that can be co-stimulatory. This will help to definewhether BTL-II can alter co-stimulation seen with other molecules.Further variations can include use of non-irradiated antigenpresentation cells of various sorts. This will help define the effectsof addition of a soluble BTL-II protein on the function of antigenpresenting cells. In still another variation, antibodies that blockBTL-II binding to cells (see above) can also be introduced in suchcostimulation experiments to determine the effects of preventingbinding.

In another kind of experiment, antigen presenting cells (such as, forexample, dendritic cells or B cells from Peyer's patches or intestinalepithelial cells (IECs)) that express BTL-II can be combined with Tcells (such as the various kinds listed above), and one or more of theparameters listed above can be measured. The results obtained with thesecells can be compared to results obtained when interfering RNAs, or DNAsencoding them, designed to lower BTL-II expression are introduced intothe antigen presenting cells.

T regulatory cells act to suppress autoimmune responses and help to doso, in part, by inducing differentiation or enhancing regulatoryfunction of T regulatory (T reg) cells in inflammatory bowel diseasemodel systems. T reg cells include, for example, Tr1 cells (Cong et al.(2002), J. Immunol. 169(11): 6112-19; Groux et al. (1997), Nature 389:737-42), Th3 cells, CD4⁻Cd25⁺ T reg cells (see e.g. Maloy and Powrie(2001), Nature Immunology 2(9): 816-22), CD4⁻Rb^(lo)CD25⁺ T reg cells,and CD8⁺ T reg cells (Allez et al. (2002), Gastroenterology 123:1516-26), among others. Thus, assessing the effects of BTL-II upon Tregulatory cell proliferation and function may be necessary to determinethe precise role of BTL-II in vivo. For example, antigen specificproliferation of T cells and/or cytokine production by T cells in thepresence of Tr1 cells can be measured. Such assays are described in Conget al., supra. Soluble forms of BTL-II, blocking antibodies, orinhibition of BTL-II expression using interfering RNAs can be used todetermine whether BTL-II plays a role in T regulatory cell or Tr1 cellproliferation, maintenance, or ability to suppress antigen activation ofother T cells.

One T cell subset, Th3 cells, produce transforming growth factor β (TGFβ) and have been implicated in oral tolerance to mucosal antigens. Seee.g. Fukaura et al. (1996), J. Clin. Invest. 98(1): 70-77; Inobe et al.(1998), Eur. J. Immunol. 28: 2780-90. Such cells can be generated fromnaïve T cells by extensive culture with antigen in the presence of TGFβ. Soluble BTL-II proteins or blocking antibodies can be used to askwhether BTL-II can alter the proliferation or function of these cells bymethods similar to those described above.

The effects of administering soluble BTL-II proteins or antagonisticantibodies in model T cell systems using simple, well-defined antigens,such as, for example, ovalbumin, or complex antigens, including bacteriaor viruses, will be ascertained. T cells responses to such antigens(including proliferation and/or production of molecules such asinterferon γ or IL-2) can be measured in the presence and absence ofBTL-II proteins or antibodies.

Similarly effects in animal systems can be tested. Early focus will beon systems particularly relevant to mucosal immune response. Thisincludes model systems in which antigen is fed to animals (see e.g.Yamashiro et al. (1994), Acta Paediatr. Jpn. 36: 550-56; Jain et al.(1996), Vaccine 14(13):1291-97; Chen et al. (2002), Immunology 105:171-80), inflammatory bowel disease systems such as in the dextransulfate sodium-induced inflammatory bowel disease model (Cooper et al.(1993), Lab. Invest. 69(2): 238-49; Tokoi et al. (1996), J.Gastroenterol. 31(2):182-88), the CD45RB^(hi)/CD4⁺ T cell-inducedwasting disease model (Morrissey et al. (1993), J. Exp. Med. 178:237-44), and/or model asthma systems in which antigen is fed orinstilled into the airways to provoke an immune response such as themurine ovalalbumin-induced asthma model (Brusselle et al. (1994), Clin.Exp. Allergy 24(1): 73-80). In particular, the magnitude of the humoralor cell mediated response will be measured, and the type ofimmunomodulatory cytokines produced will be measured. Such experimentscan reveal whether increased or decreased BTL-II can be of benefit indampening responses to antigens, including autoantigens, or increasingresponse to alloantigens. Effects of BTL-II proteins or anti-BTL-IIantibodies can also be ascertained in other models of inflammatorydiseases.

Assays for T-cell or thymocyte proliferation include without limitationthose described in: Current Protocols in Immunology, Coligan et al. eds,Greene Publishing Associates and Wiley-Interscience (pp. 3.1-3.19: Invitro assays for mouse lymphocyte function; Chapter 7: Immunologicstudies in humans); Takai et al. (1986), J. Immunol. 137: 3494-3500;Bertagnolli et al. (1990), J. Immunol. 145:1706-1712; Bertagnolli, etal. (1992), J. Immunol. 149: 3778-3783; Bowman et al. (1994), J.Immunol. 152: 1756-1761.

Assays for cytokine production and/or proliferation of spleen cells,lymph node cells or thymocytes include, without limitation, thosedescribed in: Kruisbeek and Shevach (1994), Polyclonal T cellstimulation, in Current Protocols in Immunology, Coligan et al. eds. Vol1 pp. 3.12.1-3.12.14, John Wiley and Sons, Toronto (1991); andSchreiber, (1994), Measurement of mouse and human interferon gamma inCurrent Protocols in Immunology, Coligan et al. eds. Vol 1 pp.6.8.1-6.8.8, John Wiley and Sons, Toronto (1991).

Assays for proliferation and differentiation of hematopoietic andlymphopoietic cells include, without limitation, those described in:Bottomly et al., 1991, Measurement of human and murine interleukin 2 andinterleukin 4, in Current Protocols in Immunology, Coligan et al. eds.Vol 1 pp. 6.3.1-6.3.12, John Wiley and Sons, Toronto (1991); deVries etal. (1991), J. Exp. Med. 173: 1205-1211; Moreau et al. (1988), Nature336:690-692; Greenberger et al. (1983), Proc Natl Acad Sci. USA 80:2931-2938; Nordan, Measurement of mouse and human interleukin 6, inCurrent Protocols in Immunology, Coligan et al. eds. Vol 1 pp.6.6.1-6.6.5, John Wiley and Sons, Toronto (1991); Smith et al., (1986),Proc Natl Acad Sci USA 83:1857-1861; Bennett et al., 1991, Measurementof human interleukin 11, in Current Protocols in Immunology Coligan etal. eds. Vol 1 pp. 6.15.1 John Wiley and Sons, Toronto (1991); Ciarlettaet al., Measurement of mouse and human Interleukin 9, in CurrentProtocols in Immunology Coligan et al. eds. Vol 1 pp. 6.13.1, John Wileyand Sons, Toronto (1991).

Assays for T-cell clone responses to antigens (which will identify,among others, polypeptides that affect APC-T cell interactions as wellas direct T-cell effects by measuring proliferation and cytokineproduction) include, without limitation, those described in: CurrentProtocols in Immunology, Coligan et al. eds, Greene PublishingAssociates and Wiley-Interscience (Chapter 3: In vitro assays for mouselymphocyte function; Chapter 6: Cytokines and their cellular receptors;Chapter 7: Immunologic studies in humans)(1991); Weinberger et al.(1980), Proc Natl Acad Sci USA 77:6091-6095; Takai et al. (1986), J.Immunol. 137:3494-3500; Takai et al. (1988), J. Immunol. 140: 508-512.

Assays for thymocyte or splenocyte cytotoxicity include, withoutlimitation, those described in: Current Protocols in Immunology, Coliganet al. eds, Greene Publishing Associates and Wiley-Interscience (Chapter3, In Vitro assays for Mouse Lymphocyte Function 3.1-3.19; Chapter 7,Immunologic studies in Humans) (1991); Herrmann and Mescher (1981),Proc. Natl. Acad. Sci. USA 78: 2488-2492; Herrmann et al. (1982), J.Immunol. 128: 1968-1974; Handa et al. (1985), J. Immunol. 135:1564-1572;Takai et al. (1986), J. Immunol. 137: 3494-3500; Takai et al. (1988), J.Immunol. 140:508-512,; Brown et al. (1994), J. Immunol. 153:3079-3092.

Assays for immunoglobulin responses and isotype switching by B cells(which will identify, among others, polypeptides that modulate T-celldependent antibody responses and that affect Th1/Th2 profiles) include,without limitation, those described in: Maliszewski (1990), J. Immunol144: 3028-3033; and Mond and Brunswick, Assays for B cell function: invitro antibody production, in Current Protocols in Immunology Coligan etal. eds. Vol 1 pp. 3.8.1-3.8.16, John Wiley and Sons, Toronto (1994).

Mixed lymphocyte reaction (MLR) assays (which will identify, amongothers, polypeptides that generate predominantly Th1 and CTL responses)include, without limitation, those described in: Current Protocols inImmunology, Coligan et al. eds, Greene Publishing Associates andWiley-Interscience (Chapter 3, In Vitro assays for Mouse LymphocyteFunction 3.1-3.19; Chapter 7, Immunologic studies in Humans); Takai etal. (1986), J. Immunol. 137:3494-3500; Takai et al. (1988), J. Immunol.140:508-512; Bertagnolli et al. (1992), J. Immunol. 149: 3778-3783.

Dendritic cell-dependent assays (which will identify, among others,polypeptides expressed by dendritic cells that activate naive T-cells)include, without limitation, those described in: Guery and Adorini(1995), J. Immunol 154: 536-544; Inaba et al. (1991), J Exp Med 173:549-559; Macatonia et al. (1995), J Immunol 154:5071-5079; Porgador andGilboa (1995), J Exp Med 182: 255-260; Nair et al. (1993), J. Virology67:4062-4069; Huang et al. (1994), Science 264:961-965; Macatonia et al.(1989), J Exp Med 169:1255-1264; Bhardwaj et al. (1994), J. Clin.Invest. 94:797-807; and Inaba et al. (1990), J Exp Med 172:631-640.

Assays for polypeptides that influence early steps of T-cell commitmentand development include, without limitation, those described in: Anticaet al. (1994), Blood 84: 111-117; Fine et al. (1994), Cell Immunol 155:111-122; Galy et al. (1995), Blood 85: 2770-2778; Toki et al. (1991),Proc Natl Acad Sci. USA 88: 7548-7551.

Assays for receptor-ligand activity include without limitation thosedescribed in: Current Protocols in Immunology Coligan et al. eds, GreenePublishing Associates and Wiley-Interscience (Chapter 7.28, Measurementof cellular adhesion under static conditions 7.28.1-7.28.22); Takai etal. (1987), Proc. Natl. Acad. Sci. USA 84:6864-6868; Bierer et al.(1988), J. Exp. Med. 168:1145-1156; Rosenstein et al. (1989), J. Exp.Med. 169: 149-160; Stoltenborg et al. (1994), J. Immunol. Methods 175:59-68; Stitt et al. (1995), Cell 80: 661-670.

Genetic Assay for Function Utilizing Transgenic Animals

Transgenic animals, preferably mice, that have multiple copies of thegene(s) corresponding to the BTL-II nucleic acids disclosed herein,preferably produced by transformation of cells with genetic constructsthat are stably maintained within the transformed cells and theirprogeny, are provided. Transgenic animals that have modified geneticcontrol regions that increase or reduce gene expression levels, or thatchange temporal or spatial patterns of gene expression, are alsoprovided (see European Patent No. 0 649 464 B1). In addition, organismsare provided in which the BTL-II gene has been partially or completelyinactivated, through insertion of extraneous sequences into thecorresponding gene or through deletion of all or part of thecorresponding gene. Partial or complete gene inactivation can beaccomplished through insertion, preferably followed by impreciseexcision, of transposable elements (Plasterk (1992), Bioessays 14(9):629-633; Zwaal et al. (1993), Proc. Natl. Acad. Sci. USA 90(16):7431-7435; Clark et al., (1994), Proc. Natl. Acad. Sci. USA 91(2):719-722), or through homologous recombination, preferably detected bypositive/negative genetic selection strategies (Mansour et al. (1988),Nature 336:348-352; U.S. Pat. Nos. 5,464,764; 5,487,992; 5,627,059;5,631,153; 5,614,396; 5,616,491; and 5,679,523). As an alternative,expression of the BTL-II gene can be inhibited in a transgenic ornon-transgenic animal by introduction of an interfering RNA, antisenseRNA, or a ribozyme, which may be encoded by DNA introduced into atransgenic animal. The phenotypes of such organisms can elucidate the invivo function(s) of the BTL-II gene. For example, an increasedpropensity (compared to wild type animals) in a transgenic animal thatdoes not express BTL-II protein to exhibit symptoms of inflammatorybowel disease in response to feeding of dextran sulfate sodium canindicate that BTL-II normally dampens inflammation in the gut.Alternatively, transgenic animals can overexpress BTL-II. Phenotypes ofsuch transgenic animals can also give clues as to the in vivo functionof BTL-II proteins.

Uses of the Proteins, Antibodies, Antagonists, and Agonists of theInvention

The mucosal immune system operates within a set of specializedanatomical structures, the mucus membranes, and the immune response itgenerates has properties that distinguish it from an immune responsegenerated in other anatomical compartments of the body. The mucusmembranes are just one of several distinct anatomical compartments, alsoincluding the peripheral lymph nodes and spleen, the body cavities, i.e.the peritoneum and the pleura, and the skin, in which the immune systemis active. Mucosal surfaces are found in the lungs, the gut, the eyes,the nose, the mouth, the throat, the uterus, and the vagina. A mucosalsurface is a thin sheet or layer of pliable tissue serving as thecovering or envelope of a bodily structure, such as the lining of a bodycavity, a partition or septum, or a connection between two structures.Since mucosal surfaces are the route of entry for the vast majority ofinfectious agents, an adjuvant that can promote a mucosal immuneresponse is particularly desirable. Janeway et al., Immunobiology: TheImmune System in Health and Disease, 5^(th) Edition, Part IV, Ch. 10,Garland Publishing, New York and London (2001). Infectious diseases thattypically enter through mucosal surfaces, such as, for example,Neisseria gonorrhoeae, often generate only a weak mucosal immuneresponse. Russell et al. (1999), 42(1): 58-63.

The gut is unique in several ways. Lining the gut are a number ofspecialized forms of lymphoid tissue collectively known asgut-associated lymphoid tissue (GALT) including: tonsils and adenoids,which together form Waldyer's ring at the back of the mouth; Peyer'spatches in the small intestine; the appendix; and solitary lymphoidfollicles in the large intestine and rectum. Activation of a lymphocyteby an encounter with a foreign antigen in the gut can lead to the spreadof an adaptive immune response to the antigen throughout the mucosalimmune system. The activated lymphocyte can enter the lymphatic systemand, from there, the bloodstream. The bloodstream can deliver activatedlymphocytes to mucosal sites throughout the body, which can berecognized by the lymphocytes by means of molecules such as the mucosaladressin MAdCAM-1, which is expressed in mucosal tissue. Janeway et al.,supra. The dominant antibody type produced by the gut is IgA, which,upon expression, is found primarily in the mucus layer overlying the gutepithelium. In addition, a number of distinct kinds of T cells are foundin the gut. Janeway et al., supra.

Introduction of a foreign antigen into the gut usually leads toimmunological tolerance but may lead to a specific immune response. Thegut normally receives and tolerates (in an immunological sense) a vastarray of foreign antigens, that is, food and the commensalmicroorganisms residing in the gut. The feeding of a specific foreignprotein can lead to a state of specific unresponsiveness to that proteinknown as oral tolerance, such that later injection of the protein, evenin the presence of an adjuvant, yields no antibody response. Thisphenomenon may involve the spleen and lymph nodes as well as the mucosalimmune system. See Gutgemann et al. (1998), Immunity 8: 667-73. However,enteric pathogens, such as, for example, Salmonella, Yersinia, orEntamoeba histolytica, can elicit a local, or even a systemic, immuneresponse. The factors controlling whether the introduction of a foreignantigen into the gut elicits an immune response are incompletelyunderstood. Janeway et al., supra at Part V, Ch. 14.

Many vaccines employ adjuvants to enhance the immune response to theantigen. An adjuvant strengthens or broadens the specificity of animmune response to an antigen. The immune response may include anincrease in antibody titer or an increase in the number ofantigen-reactive T cells. Methods for measuring such parameters exist inthe art. See e.g. Zigterman et al. (1988), J. Immunol. Methods 106(1):101-07. The mechanism(s) by which adjuvants enhance an immune responseare incompletely understood, but their use can be essential. Fewexisting vaccines can elicit a robust mucosal immune response to theselected antigen.

The invention encompasses a method for promoting a systemic or mucosalimmune response against an antigen comprising administering atherapeutically effective amount of an antagonist of BTL-II and theantigen. The antagonists of BTL-II that can be used to practice theinvention include, antagonistic antibodies or in vitro-selected bindingproteins that bind specifically to the extracellular region of BTL-II,and small molecules that can inhibit the biological activity of BTL-II.Optionally, the antagonist of BTL-II protein and the antigen can beadministered directly to a mucosal surface, such as orally, nasally,vaginally, gastrically, or rectally or by inhalation. For example, nasaladministration has been reported to be more effective than vaginaladministration in inducing a durable immune response in at least onecase. Russell (2002), Am. J. Reprod. Immunol. 47(5): 265-68.Alternatively, the BTL-II antagonist and/or the antigen can be injected,for example, subcutaneously, intravenously, intramuscularly,intraarterially, or intraperitoneally. In some embodiments, a BTL-IIantagonist, such as an antibody, can be injected and an antigen can beadministered directly to a mucosal surface.

Appropriate antigens for practicing the invention include all or part ofany infectious agent or agent that is similar to an infectious agent.Infectious agents can include live or killed viruses, bacteria, andinfectious eukaryotes such as amoeba, flagellates, or helminths. Anagent that is similar to such an infectious agent may, for example, be avirus that is analogous to a virus that can infect the mammal beingvaccinated, but cannot, itself, infect the mammal being vaccinated. Anexample of this is the vaccinia virus (which can produce disease in cowsbut not people) used by Jenner to produce a vaccine against smallpox, asimilar virus that produces disease in humans. Janeway et al., supra,Part V, Ch. 14. Table 4 indicates specific examples of antigens thatcould be used to practice the invention.

TABLE 4 Antigen Category Some Specific Examples of RepresentativeAntigens Viruses Rotavirus; foot and mouth disease; influenza, includinginfluenza A and B; parainfluenza; Herpes species (Herpes simplex,Epstein-Barr virus, chicken pox, pseudorabies, cytomegalovirus); rabies;polio; hepatitis A; hepatitis B; hepatitis C; hepatitis E; measles;distemper; Venezuelan equine encephalomyelitis; feline leukemia virus;reovirus; respiratory syncytial virus; bovine respiratory syncytialvirus; Lassa fever virus; polyoma tumor virus; parvovirus; canineparvovirus; papilloma virus; tick-borne encephalitis; rinderpest; humanrhinovirus species; enterovirus species; Mengo virus; paramyxovirus;avian infectious bronchitis virus; HTLV 1; HIV-1; HIV-2; LCMV(lymphocytic choriomeningitis virus); adenovirus; togavirus (rubella,yellow fever, dengue fever); corona virus Bacteria Bordetella pertussis;Brucella abortis; Escherichia coli; Salmonella species includingSalmonella typhi; streptococci; Vibrio species (V. cholera, V.parahaemolyticus); Shigella species; Pseudomonas species; Brucellaspecies; Mycobacteria species (tuberculosis, avium, BCG, leprosy);pneumococci; staphlylococci; Enterobacter species; Rochalimaia henselae;Pasterurella species (P. haemolytica, P. multocida); Chlamydia species(C. trachomatis, C. psittaci, Lymphogranuloma venereum); Syphilis(Treponema pallidum); Haemophilus species; Mycoplasma species; Lymedisease (Borrelia burgdorferi); Legionnaires' disease; Botulism(Colstridium botulinum); Corynebacterium diphtheriae; Yersiniaentercolitica Ricketsial Rocky mountain spotted fever; thyphus;Ehrlichia species Infections Parasites Malaria (Plasmodium falciparum,P. vivax, P. malariae); schistosomes; and trypanosomes; Leishmaniaspecies; filarial nematodes; trichomoniasis; Protozoa sarcosporidiasis;Taenia species (T. saginata, T. solium); Toxoplasma gondii;trichinelosis (Trichinella spiralis); coccidiosis (Eimeria species);helminths including Ascarus species Fungi Cryptococcus neoformans;Candida albicans; Apergillus fumigatus; coccidioidomycosis RecombinantHerpes simplex; Epstein-Barr virus; hepatitis B; pseudorabies; Proteinsflavivirus (dengue, yellow fever); Neisseria gonorrhoeae; malaria:circumsporozoite protein, merozoite protein; trypanosome surface antigenprotein; pertussis; alphaviruses; adenovirus Proteins Diphtheria toxoid;tetanus toxoid; meningococcal outer membrane protein (OMP);streptococcal M protein; hepatitis B; influenza hemagglutinin; cancerantigen; tumor antigens; toxins; exotoxins; neurotoxins; cytokines andcytokine receptors; monokines and monokine receptors Synthetic Malaria;influenza; foot and mouth disease virus; hepatitis B; hepatitis CPeptides Antigen Some Specific Examples of Representative AntigensCategory Poly- Pneumococcal polysaccharide; Haemophilis influenzapolyribosyl- saccharides ribitolphosphate (PRP); Neisseria meningitides;Pseudomonas aeruginosa; Klebsiella pneumoniae Oligo- Pneumococcalsaccharide

Alternatively, soluble BTL-II proteins can be used to promote toleranceto an antigen that is implicated in an autoimmune or inflammatorydisease. For example, experimental autoimmune encephalomyelitis (EAE), acondition similar in many respects to multiple sclerosis, can be inducedin rodents by injection of, for example, various epitopes of myelinbasic protein or myelin oligodendrocyte glycoprotein (MOG). MOG-inducedEAE can, in some cases, be ameliorated by prior feeding of smallportions of MOG or butyrophilin. Stefferl et al. (2000), J. Immunol.165:2859-65. Soluble BTL-II proteins can be co-administered with anantigen known to be targeted in an autoimmune disease to promotetolerance to the antigen and thereby ameliorate the symptoms of theautoimmune disease. Optionally, the antigen can be administered directlyto a mucosal surface, for example, nasally.

Inflammatory bowel diseases, including Crohn's disease and ulcerativecolitis, include chronic inflammation of the gastrointestinal tract,possibly because of an abnormally enhanced immune response to antigensof normal gut flora. Both diseases likely have at least some geneticbasis since occurrences tend to cluster in families and can beassociated with some genetic markers. For example, mice that do notexpress the multiple drug resistance gene (mdr1a) spontaneously developcolitis. Panwala et al. (1998), J. Immunol. 161: 5733-44. The occurrenceof both Crohn's disease and ulcerative colitis is likely also influencedby environmental factors because increased occurrence is observed amongurbanized populations. Also, such diseases do not occur in the absenceof normal gut flora.

Crohn's disease is involves an abnormal inflammation of any portion ofthe alimentary tract from the mouth to the anus, although in mostpatients abnormal inflammation is confined to the ileocolic,small-intestinal, and colonic-anorectal regions. Typically, theinflammation is discontinuous. Common symptoms include abdominal pain,anorexia, weight loss, fever, diarrhea, fullness and/or tenderness inthe right lower quadrant of the abdomen, constipation, vomiting, andperianal discomfort and discharge. Other possible symptoms includeperipheral arthritis, growth retardation, episcleritis, aphthousstomatitis, erythema nodosum, pyoderma gangrenosum, kidney stones,impaired urinary dilution and alkalinization, malabsorption, andgallstones, among others. See e.g. Strober et al., Medical Immunology,10^(th) Edition, Section III, Ch. 35 (2001); Merck Manual of Diagnosisand Therapy, 17^(th) Edition, Section 3, Ch. 31 (1999). Macrophagesisolated from patients with Crohn's disease produce increased amounts ofIL-12, IFNγ, TNFα, and other inflammatory cytokines

Ulcerative colitis, though it is sometimes hard to distinguish fromCrohn's disease, is distinct from Crohn's disease in several respects.First, it is generally limited to the colon while Crohn's disease mayoccur throughout the alimentary tract. Second, ulcerative colitis mainlyinvolves inflammation only of the superficial layers of the bowel,unlike Crohn's disease in which the inflammation can penetrate all waythrough the wall of the bowel or other location in the alimentary tract.Finally, ulcerative colitis typically involves a continuous area ofinflammation, rather than the discontinuous sites of inflammationtypical of Crohn's disease. Like Crohn's disease, ulcerative colitis isfound primarily in urban areas. Also, genetic factors likely play a rolein ulcerative colitis since there is a familial aggregation of cases.Autoantibodies are observed in ulcerative colitis patients more oftenthan Crohn's disease patients. The autoantibodies are often directed tocolonic epithelial cell components. Among the most common areantineutrophil cytoplasmic antibodies with specificities for catalase,α-enolase, and lactoferrin. In some cases such antibodies cross reactwith colonic microorganisms.

Symptoms of ulcerative colitis are variable. They may include diarrhea,tenesmus, abdominal cramps, blood and mucus in the stool, fever, andrectal bleeding. Toxic megacolon, a potentially life-threateningcondition in which the colon is dilated beyond about 6 centimeters andmay lose its muscular tone and/or perforate, may also occur. Othersymptoms that may accompany ulcerative colitis include peripheralarthritis, ankylosing spondylitis, sacroiliitis, anterior uveitis,erythema nodosum, pyoderma gangrenosum, episcleritis, autoimmunehepatitis, primary sclerosing cholangitis, cirrhosis, and retardedgrowth and development in children.

Antibodies or in vitro-selected binding proteins that bind specificallyto BTL-II proteins can be used to diagnose or predict the onset ofinflammatory bowel disease. As illustrated in Example 4 below, BTL-II isoverexpressed in the gut prior to the onset of symptoms and during thesymptomatic phase in a mouse model of inflammatory bowel disease. Thus,overexpression of BTL-II can indicate the existence of inflammatorybowel disease and can predict the its onset. Anti-BTL-II antibodies canbe used to detect overexpression of BTL-II by assaying a tissue samplefrom the bowel of a patient using an ELISA assay or other immune-basedassays known in the art. See e.g. Reen (1994), Enzyme-LinkedImmunosorbent Assay (ELISA), in Basic Protein and Peptide Protocols,Methods Mol. Biol. 32: 461-466. Overexpression can also be detected bynucleic acid-based methods for measuring BTL-II mRNA expression such as,for example, reverse transcription plus PCR (RT-PCR), among other mRNAexpression assays known in the art. See e.g. Murphy et al. (1990),Biochemistry 29(45): 10351-56.

In another embodiment, soluble BTL-II proteins of the invention can beused to treat an inflammatory bowel disease. A soluble BTL-II proteincan bind to a specific receptor expressed on a B cell or a T cell,thereby enabling the downregulation of an immune response. Such adownregulation can, for example, prevent the activation of a macrophageor a B cell by a CD4⁺ T cell or prevent activation of a T cell by anantigen. Alternatively, such a downregulation can cause a T cell tobecome anergic when it encounters an antigen to which its T cellreceptor can specifically bind.

Antibodies or in vitro-selected binding proteins that bind specificallyto BTL-II also find use as diagnostic reagents to identify patients withinflammatory bowel disease or at risk of developing inflammatory boweldisease. Since BTL-II is overexpressed prior to the onset of and duringinflammatory bowel disease symptoms in a mouse model system (Example 4),an abnormally high level of BTL-II expression can indicate the presenceof an inflammatory disease in the gut or a high risk of developing aninflammatory disease in the gut.

In addition, soluble BTL-II proteins can be useful in situations wheredown-modulation of an immune response is desired, such astransplantation (Manilay et al., 1998, Curr. Opin. Immunol. 10:532-538),graft versus host disease, graft rejection, autoimmune or inflammatorydisease, gene therapy (Hackett et al., 2000, Curr. Opin. Mol. Therap. 2:376-382), and the like. For example, a soluble BTL-II protein can beadministered prior to, at approximately the same time (or either shortlybefore or shortly after), or concurrently with administration of a genetherapy vector to a mammal, transplantation, or as otherwise appropriatefor the desired immuno-suppression. Also appropriate for such atreatment is an anti-idiotypic antibody that mimics the function ofBTL-II.

An agonistic BTL-II antibody, a soluble BTL-II protein, or ananti-idiotypic antibody can be administered to a patient suffering froman autoimmune or inflammatory disease in order to decrease the number ofdetectable autoantibodies, to decrease the activation of immune effectorcells, and/or to decrease or eliminate the symptoms of the disease.Autoimmune and inflammatory diseases include all conditions in which thepatient's own tissues are subject to deleterious effects caused by thepatient's immune system. Such effects can be mediated by autoantibodiesand/or by the activation of immune effector cells, among otherpossibilities. Although the causes of autoimmune and inflammatorydiseases are usually unclear, a correlation between the existence ofvarious kinds of infections and various autoimmune diseases has beenestablished in some cases and is a recurring subject of discussion inthe scientific literature. See e.g. Corapcioglu et al. (2002), Thyroid12:613-17;Sewell et al. (2002), Immunol. Lett. 82: 101-10; Rose (1998),Semin. Immunol. 10(1): 5-13; Matsiota-Bernard (1996), Clin. Exp.Immunol. 104: 228-35; and McMurray and Elbourne (1997), Semin. ArthritisRheum. 26: 690-701.

One of skill in the art will appreciate that symptoms of autoimmune andinflammatory diseases are extremely diverse and can depend on whattissues are targeted by the patient's immune system. Autoimmune andinflammatory diseases can be organ-specific or systemic. Autoimmune andinflammatory diseases include, for example, arthritis, Addison'sdisease, insulin-dependent diabetes mellitus (type I diabetes mellitus),asthma, polyglandular endocrinopathy syndromes, systemic lupuserythematosus, chronic active hepatitis, various forms of thyroiditis(including Hashimoto's thyroiditis, transient thyroiditis syndromes, andGrave's disease), lymphocytic adenohypophysitis, premature ovarianfailure, idiopathic phyoparathyroidism, pernicious anemia,glomerulonephritis, autoimmune neutropenia, Goodpasture's syndrome,multiple sclerosis, vitiligo, myasthenia gravis, rheumatoid arthritis,scleroderma, primary Sjogren's syndrome, polymyositis, autoimmunehemolytic anemia, inflammatory bowel disease (including Crohn's diseaseand ulcerative colitis), psoriasis, psoriatic arthritis, dermatitis,autoimmune thrombocytopenic purpura, pemphigus vulgaris, acute rheumaticfever, mixed essential cryoglobulinemia, and warm autoimmune hemolyticanemia, among many others.

Vectors and Host Cells

The present invention also provides vectors containing the nucliec acidsof the invention, as well as host cells transformed with such vectors.Any of the nucleic acids of the invention may be contained in a vector,which generally includes a selectable marker and an origin ofreplication, for propagation in a host. The vectors further includesuitable transcriptional or translational regulatory sequences, such asthose derived from a mammalian, microbial, viral, or insect genes,operably linked to the BTL-II nucleic acid. Examples of such regulatorysequences include transcriptional promoters, operators, or enhancers,mRNA ribosomal binding sites, and appropriate sequences that controltranscription and translation. Nucleotide sequences are operably linkedwhen the regulatory sequence functionally relates to the DNA encodingthe target protein. Thus, a promoter nucleotide sequence is operablylinked to a BTL-II nucleic sequence if the promoter nucleotide sequencedirects the transcription of the BTL-II sequence.

Selection of suitable vectors for the cloning of BTL-II nucleic acidsencoding the target BTL-II proteins of this invention will depend uponthe host cell in which the vector will be transformed, and, whereapplicable, the host cell from which the target polypeptide is to beexpressed. Suitable host cells for expression of BTL-II proteins includeprokaryotes, yeast, insect, and higher eukaryotic cells, each of whichis discussed below.

The BTL-II proteins to be expressed in such host cells may also befusion proteins that include regions from heterologous proteins. Asdiscussed above, such regions may be included to allow, for example,secretion, improved stability, facilitated purification, targeting, oroligomerization of the BTL-II protein. For example, a nucleic acidsequence encoding an appropriate signal sequence can be incorporatedinto an expression vector. A nucleic acid sequence encoding a signalsequence (secretory leader) may be fused in-frame to a BTL-II sequenceso that BTL-II is translated as a fusion protein comprising the signalpeptide. A signal peptide can be functional in the intended host cellcan promote extracellular secretion of the BTL-II protein. Aheterologous signal peptide can replace the native signal sequence.Examples of signal peptides that are functional in mammalian host cellsinclude the signal sequence for interleukin-7 (IL-7) described in U.S.Pat. No. 4,965,195, the signal sequence for interleukin-2 receptordescribed in Cosman et al. ((1984), Nature 312:768); the interleukin-4receptor signal peptide described in EP Patent No. 0 367 566; the type Iinterleukin-1 receptor signal peptide described in U.S. Pat. No.4,968,607; the type II interleukin-1 receptor signal peptide describedin EP Patent No. 0 460 846; the signal sequence of human IgK (which isMETDTLLLWVLLLWVPGSTG; SEQ ID NO:29); and the signal sequence of humangrowth hormone (which is MATGSRTSLLLAFGLLCLPWLQEGSA; SEQ ID NO:30).Preferably, the signal sequence will be cleaved from the BTL-II proteinupon secretion of the BTL-II protein from the cell. Other signalsequences that can be used in practicing the invention include the yeasta-factor and the honeybee melatin leader in Sf9 insect cells. Brake(1989), Biotechnology 13: 269-280; Homa et al. (1995), Protein Exp.Purif. 6141-148; Reavy et zal. (2000), Protein Exp. Purif. 6:221-228.

Suitable host cells for expression of the proteins of the inventioninclude prokaryotes, yeast, and higher eukaryotic cells. Suitableprokaryotic hosts to be used for the expression of these polypeptidesinclude bacteria of the genera Escherichia, Bacillus, and Salmonella, aswell as members of the genera Pseudomonas, Streptomyces, andStaphylococcus. For expression in prokaryotic cells, for example, in E.coli, the polynucleotide molecule encoding BTL-II protein preferablyincludes an N-terminal methionine residue to facilitate expression ofthe recombinant polypeptide. The N-terminal Met may optionally becleaved from the expressed polypeptide.

Expression vectors for use in cellular hosts generally comprise one ormore phenotypic selectable marker genes. Such genes encode, for example,a protein that confers antibiotic resistance or that supplies anauxotrophic requirement. A wide variety of such vectors are readilyavailable from commercial sources. Examples include pGEM vectors(Promega), pSPORT vectors, and pPROEX vectors (InVitrogen, LifeTechnologies, Carlsbad, Calif.), Bluescript vectors (Stratagene), andpQE vectors (Qiagen).

BTL-II can also be expressed in yeast host cells from genera includingSaccharomyces, Pichia, and Kluveromyces. Preferred yeast hosts are S.cerevisiae and P. pastoris. Yeast vectors will often contain an originof replication sequence from a 2μ yeast plasmid, an autonomouslyreplicating sequence (ARS), a promoter region, sequences forpolyadenylation, sequences for transcription termination, and aselectable marker gene. Vectors replicable in both yeast and E. coli(termed shuttle vectors) may also be used. In addition to theabove-mentioned features of yeast vectors, a shuttle vector will alsoinclude sequences for replication and selection in E. coli. Directsecretion of the target polypeptides expressed in yeast hosts may beaccomplished by the inclusion of nucleotide sequence encoding the yeasta-factor leader sequence at the 5′ end of the BTL-II-encoding nucleotidesequence. Brake (1989), Biotechnology 13:269-280.

Insect host cell culture systems can also be used for the expression ofBTL-II proteins. The proteins of the invention are preferably expressedusing a baculovirus expression system, as described, for example, in thereview by Luckow and Summers ((1988), BioTechnology 6: 47).

BTL-II proteins of the invention can be expressed in mammalian hostcells. Non-limiting examples of suitable mammalian host cell linesinclude the COS-7 line of monkey kidney cells (Gluzman et al. (1981),Cell 23: 175-182), Chinese hamster ovary (CHO) cells (Puck et al.(1958), PNAS USA 60: 1275-1281), CV-1 (Fischer et al. (1970), Int. J.Cancer 5: 21-27) and human cervical carcinoma cells (HELA) (ATCC CCL 2).

The choice of a suitable expression vector for expression of BTL-IIproteins of the invention will depend upon the specific mammalian hostcell to be used. Examples of suitable expression vectors includepcDNA3.1/Hygro⁺ (Invitrogen), pDC409 (McMahan et al. (1991), EMBO J. 10:2821-2832), and pSVL (Pharmacia Biotech). Expression vectors for use inmammalian host cells can include transcriptional and translationalcontrol sequences derived from viral genomes. Commonly used promotersequences and enhancer sequences that can be used to express BTL-IIinclude, but are not limited to, those derived from humancytomegalovirus (CMV), Adenovirus 2, Polyoma virus, and Simian virus 40(SV40). Methods for the construction of mammalian expression vectors aredisclosed, for example, in Okayama and Berg ((1982) Mol. Cell. Biol.2:161-170), Cosman et al. ((1986) Mol. Immunol. 23:935-941), Cosman etal. ((1984) Nature 312: 768-771), EP-A-0367566, and WO 91/18982.

Modification of a BTL-II nucleic acid molecule to facilitate insertioninto a particular vector (for example, by modifiying restriction sites),ease of use in a particular expression system or host (for example,using preferred host codons), and the like, are known and arecontemplated for use in the invention. Genetic engineering methods forthe production of BTL-II proteins include the expression of thepolynucleotide molecules in cell free expression systems, in cellularhosts, in tissues, and in animal models, according to known methods.

Therapeutic Methods

“Treatment” of any disease mentioned herein encompasses an alleviationof at least one symptom of the disease, a reduction in the severity ofthe disease, or the delay or prevention of disease progression to moreserious symptoms that may, in some cases, accompany the disease or to atleast one other disease. Treatment need not mean that the disease istotally cured. A useful therapeutic agent needs only to reduce theseverity of a disease, reduce the severity of symptom(s) associated withthe disease or its treatment, or delay the onset of more serioussymptoms or a more serious disease that can occur with some frequencyfollowing the treated condition. For example, if the disease is aninflammatory bowel disease, a therapeutic agent may reduce the number ofdistinct sites of inflammation in the gut, the total extent of the gutaffected, reduce pain and/or swelling, reduce symptoms such as diarrhea,constipation, or vomiting, and/or prevent perforation of the gut. Apatient's condition can be assessed by standard techniques such as anx-ray performed following a barium enema or enteroclysis, endoscopy,colonoscopy, and/or a biopsy. Suitable procedures vary according to thepatient's condition and symptoms.

The invention encompasses a method of treating inflammatory diseases,including autoimmune diseases, graft versus host disease, andinflammatory bowel diseases, using an amount of a BTL-II protein orantibody for a time sufficient to induce a sustained improvement overbaseline of an indicator that reflects the severity of a particulardisorder or the severity of symptoms caused by the disorder or to delayor prevent the onset of a more serious disease that follows the treatedcondition in some or all cases. The treatments of the invention may beused before, after, or during other treatments for the disorder inquestion that are commonly used, or they may be used without othertreatments. For example, Crohn's disease and ulcerative colitis arecommonly treated with sulfasalazine, 5-aminosalicylic acid, orcortico-steroids. These treatments may be used before, during, or afterthe treatments of the invention.

Any of the above-described therapeutic agents can be administered in theform of a composition, that is, with one or more additional componentssuch as a physiologically acceptable carrier, excipient, or diluent. Forexample, a composition may comprise a soluble BTL-II protein asdescribed herein plus a buffer, an antioxidant such as ascorbic acid, alow molecular weight polypeptide (such as those having less than 10amino acids), a protein, amino acids, carbohydrates such as glucose,sucrose, or dextrins, chelating agent such as EDTA, glutathione, and/orother stabilizers, excipients, and/or preservatives. The composition maybe formulated as a liquid or a lyophilizate. Further examples ofcomponents that may be employed in pharmaceutical formulations arepresented in Remington's Pharmaceutical Sciences, 16^(th) Ed., MackPublishing Company, Easton, Pa., (1980).

Compositions comprising therapeutic molecules described above can beadministered by any appropriate means including, but not limited to,parenteral, topical, oral, nasal, vaginal, rectal, or pulmonary (byinhalation) administration. If injected, the composition(s) can beadministered intra-articularly, intravenously, intraarterially,intramuscularly, intraperitoneally, or subcutaneously by bolus injectionor continuous infusion. Localized administration, that is, at the siteof disease, is contemplated, as are transdermal delivery and sustainedrelease from implants, skin patches, or suppositories. Delivery byinhalation includes, for example, nasal or oral inhalation, use of anebulizer, inhalation in aerosol form, and the like. Administration viaa suppository inserted into a body cavity can be accomplished, forexample, by inserting a solid form of the composition in a chosen bodycavity and allowing it to dissolve. In the case of soluble BTL-IIproteins or agonists to treat an inflammatory bowel disease,administration via a rectal suppository may be particularly appropriatesince it localizes the therapeutic appropriately. Other alternativesinclude eyedrops, oral preparations such as pills, lozenges, syrups, andchewing gum, and topical preparations such as lotions, gels, sprays, andointments. In most cases, therapeutic molecules that are polypeptidescan be administered topically or by injection or inhalation.

The therapeutic molecules described above can be administered at anydosage, frequency, and duration that can be effective to treat thecondition being treated. The dosage depends on the molecular nature ofthe therapeutic molecule and the nature of the disorder being treated.Treatment may be continued as long as necessary to achieve the desiredresults. Therapeutic molecules of the invention can be administered as asingle dosage or as a series of dosages given periodically, includingmultiple times per day, daily, every other day, twice a week, threetimes per week, weekly, every other week, and monthly dosages, amongother possible dosage regimens. The periodicity of treatment may or maynot be constant throughout the duration of the treatment. For example,treatment may initially occur at weekly intervals and later occur everyother week. Treatments having durations of days, weeks, months, or yearsare encompassed by the invention. Treatment may be discontinued and thenrestarted. Maintenance doses may be administered after an initialtreatment.

Dosage may be measured as milligrams per kilogram of body weight (mg/kg)or as milligrams per square meter of skin surface (mg/m²) or as a fixeddose, irrespective of height or weight. All of these are standard dosageunits in the art. A person's skin surface area is calculated from herheight and weight using a standard formula.

The invention has been described with reference to specific examples.These examples are not meant to limit the invention in any way. It isunderstood for purposes of this disclosure, that various changes andmodifications may be made to the invention that are well within thescope of the invention. Numerous other changes may be made which willreadily suggest themselves to those skilled in the art and which areencompassed in the spirit of the invention disclosed herein and asdefined in the appended claims.

This specification contains numerous citations to patents, patentapplications, and publications. Each is hereby incorporated by referencefor all purposes.

Example 1 Isolation of Human BTL-II cDNAs

RNA was isolated from several sources, human colon tissue samples frompatients with Crohn's disease or ulcerative colitis, the human coloncancer cell line Caco-2 (American Type Culture Collection (ATCC) No.HTB-37), and a colon epithelial cell line called T84 (ATCC No. CCL-248).The RNA was reverse transcribed and amplified by PCR using primers thatwere designed on the basis of the nucleic acid sequence disclosed inNCBI accession no. NM_(—)019602. This yielded an upstream portion of thesequences of full length BTL-II (SEQ ID NO:3) and the splice variantsequences disclosed in SEQ ID NO:7, SEQ ID NO:9, SEQ ID NO:11, SEQ IDNO:13, and SEQ ID NO:15. Isolation of a cDNA containing the 3′ end ofthe BTL-II mRNA was accomplished using 3′ RACE (Rapid Amplification ofcDNA Ends), i.e., essentially the protocols of Frohman et al. ((1988),Proc. Natl. Acad. Sci. USA 85(23): 8998-9002). From the analysis of manyvariants of the BTL-II cDNA by 3′

RACE revealed no variants that encoded soluble proteins lacking atransmembrane domain. As shown in FIGS. 5 a, 6 a, and 7 a, many of thevariants contained sequence polymorphisms (or allelic variations) at anumber of sites in the BTL-II sequence.

Example 2 Isolation of Murine BTL-II cDNAs

Murine BTL-II cDNAs were isolated as follows. RNA was isolated frommurine colon and small intestine using a kit for the isolation andpurification of RNA (the RNEASY® kit; Qiagen) and treated with DNAse I(Ambion), according to recommendations of the manufacturer, to eliminateresidual chromosomal DNA. Purified RNA was transcribed into cDNA, usinga reaction mixture containing isolated RNA in 10 mM Tris-HCl, pH 8.3, 50mM KCL, 5 mM MgCl₂, 1 mM of each dNTP, 2.5 μM random hexamer primers, 1U/μl RNAse inhibitor, and 2.5 U/μl MuLV Reverse Transcriptase (PEBiosystems). The reaction mixture was incubated for 10 minutes at 25°C., followed by 30 minutes at 48° C., followed by 5 minutes at 95° C.PCR amplification reactions for the BTL-II gene were performed in afinal volume of 100 μl containing 10 mM Tris-HCL, pH 8.3, 50 mM KCl, 1.5mM MgCl₂, 200 μM of each dNTP, and 2.5U ampliTaq DNA polymerase (PerkinElmer) and 25 pmol of both the upstream (5′-TTACTGAGAGAGGGAAACGGGCTGTTTTCTCC; SEQ ID NO:31) and downstream(5′GGACTTCATTGGTGACTGATGCCATCCAC; SEQ ID NO:32) primers. Theamplification reactions were carried out for 35 cycles of 40 seconds at94° C., 40 seconds at 55° C., and 40 seconds at 72° C. The amplificationproducts were analyzed on a 2% agarose gel, visualized by ethidiumbromide, and sequenced. By visual estimation, splice variants lackingexon 3 (FIG. 9 a) were more abundant than variants containing exons 1 to8 (FIG. 8).

Example 3 Expression of BTL-II in Cells and Tissues from Various Sources

Expression of BTL-II mRNA was measured using real time PCR essentiallyaccording to the protocols of Heid et al. ((1996), Genome Res. 6(10):986-94) and using standard reverse transcription followed by PCR(RT-PCR; see e.g. Fuqua et al. (1990), Biotechniques 9(2): 206-11). Allcells types tested were of human origin except the CD11c⁺ CD8⁺ B220⁺ (orplasmacytoid) dendritic cells from Peyer's patches which were frommouse. Cells in which BTL-II expression was detected were the following:human B cells, unstimulated or stimulated with killed Staphylococcusaureus, CD40 ligand, and interleukin 4; normal human bronchialepithelial cells (NHBE cells; see Lechner et al. (1983), Cancer Res. 43(12 pt. 1): 5915-21) stimulated with interferon γ; Calu-3 cells, a lungepithelial cell line (ATCC No. HTB-55), unstimulated; T84 cells, a humancolon epithelial cell line, unstimulated or stimulated with interferonγ; Caco-2 cells, a human colon cancer cell line, unstimulated; CD11c⁻(low expressing) CD8⁺ B220⁺ cells from murine Peyer's patches, which arepredominantly dendritic cells; and murine peripheral blood leukocytes.Expression, on an absolute scale, was low in most cells tested.Expression of BTL-II mRNA was not detected in dendritic cells resultingfrom in vitro treatment of human peripheral blood monocytes to inducedifferentiation into a dendritic cell type. However, BTL-II expressionwas detected in CD123⁺ plasmacytoid dendritic cells purified from humanblood purified from human peripheral blood. BTL-II mRNA was highlyenriched in the CD11c⁺ (low expressing) CD8⁺ B220⁺ subset of murinedendritic cells from Peyer's patches. Expression of BTL-II mRNA wasdetected in a number of murine tissue types by similar methods includingspleen, lymph node, stomach, mesenteric lymph nodes, bone marrow, smallintestine, cecum, lung, large intestine, Peyer's patch, and thymus. Thehighest levels of expression were detected in small intestine, Peyer'spatch, and cecum tissue.

Example 4 Expression of BTL-II in a Murine Model for Inflammatory BowelDisease

Mdr1a−/−mice can be a model system for the study of chronic inflammatorybowel disease. Panwala et al. (1998), J. Immunol. 161: 5733-44. Themurine multiple drug resistance gene, mdr1a, encodes a 170 kDatransmembrane protein that is expressed in many tissues includingintestinal epithelial cells and lymphoid cells. Mice deficient in mdr1aare susceptible to developing severe spontaneous intestinal inflammationcharacterized by dysregulated epithelial cell growth and massiveleukocyte infiltration into the lamina propria of the large intestine.Treating mdr1a−/−mice with oral antibiotics prevents both thedevelopment of disease and resolves active inflammation. Lymphoid cellsisolated from mice with active colitis demonstrate enhanced reactivityto intestinal bacterial antigens. Although mdr1a is expressed by bothepithelial cells and leukocytes, the development of colitis correlateswith lack of mdr1a expression on epithelial cells.

The mdr1a−/− mice used were in an FVB genetic background. Typically,approximately 20% of a group of mdr1a−/−mice spontaneously developcolitis at 18 to 20 weeks of age, while the remaining mice in themdr1a−/−colony remain healthy and do not develop colitis. The percentageof animals that develop disease is dependent on the cleanliness of theanimal facility.

Initially, RNA was prepared from gut tissue and, after reversetranscription incorporating a fluorescent label into the resulting cDNA,used to hybridize to an array of probes on a custom-prepared Affymetrixchip containing an oligonucleotide designed to detect BTL-II mRNA basedon the sequence published by Stammers et al. ((2000), Immunogenetics 51:373-82). Overexpression of BTL-II mRNA was detected in mdr1a−/−mice(relative to wild type individuals of the FVB strain) both before theonset of inflammatory bowel disease symptoms and during the symptoms. Asample of such data is shown in FIG. 10. These data show that BTL-IImRNA is expressed to a greater extent in mdr1a−/−mice than in wild typeFVB mice. Moreover, even higher expression of BTL-II mRNA accompaniesthe onset of symptoms of inflammatory bowel disease.

These results were confirmed by analysis of the same RNA using a realtime PCR technique (Heid et al. (1996), Genome Res. 6(10): 986-94). Thisanalysis showed overexpression of BTL-II mRNA by two symptomaticmdr1a−/−mice relative to two healthy wild type FVB mice. This data isdiagrammed in FIG. 11. The two parental mice are represented by the twobars marked with diagonal stripes towards the left of FIG. 11, and thetwo symptomatic mdr1a−/−are represented by the two bars marked withcheckered patterns towards the right of FIG. 11. These data indicatethat there is approximately a 2 to 5 fold difference in expressionbetween the wild type mice and the symptomatic mdr1a−/−mice. Thus,higher levels of BTL-II mRNA are expressed in mice with symptoms ofinflammatory bowel disease than in wild type mice with no symptoms.

Example 5 Construction of a BTL-II:Fc Fusion Protein

A soluble BTL-II protein consisting of the extracellular region ofmurine BTL-II fused to a human Fc region (BTL-II:Fc) was produced in thefollowing way. A fusion cDNA construct encoding BTL-II:Fc was preparedby fusing nucleic acids encoding the extracellular region of murineBTL-II to nucleic acids encoding a human IgG1 (in-frame). To produce theBTL-II:Fc protein, mammalian cells were transfected with the fusion cDNAconstruct using the LIPOFECTAMINE™ 2000 transfection method (Invitrogen,Carlsbad, Calif., USA). BTL-II:Fc protein-containing supernatants wereharvested 6-7 days post transfection, and the BTL-II:Fc protein waspurified by Protein A column chromatography. The nucleic acid sequenceencoding the BTL-II:Fc protein and the BTL-II:Fc amino acid sequence arefound in SEQ ID NO:19 and SEQ ID NO:20, respectively.

Example 6 Suppression of Human T Cell Proliferation by BTL-II:Fc

The following experiment tests whether a soluble form of BTL-II cansuppress T cell proliferation in vitro in response to a monoclonalanti-CD3ε antibody with or without other costimulatory molecules.

BTL-II:Fc was made as described in Example 5. A 96 well, U bottommicrotiter plate was coated with varying concentrations of anti-CD3εantibody with or without one or more other proteins. FIG. 12 indicateswhat proteins were used in each sample as follows: anti-CD3ε antibodyalone,

; anti-CD3ε antibody and BTL-II:Fc,

; anti-CD3ε antibody and B7RP-1:Fc,

; anti-CD3ε antibody, B7RP-1:Fc, and BTL-II:Fc,

; anti-CD3ε antibody and B7-2:Fc,

; anti-CD3ε antibody, B7-2:Fc, and BTL-II:Fc,

. B7RP-1:Fc consists of the extracellular region of B7RP-1 (also knownas B7h) fused to an Fc region of an antibody, and B7-2:Fc consists ofthe extracellular region of B7-2 fused to an Fc region of an antibody.Both of these proteins are members of the B7 family of proteins known tomodulate T cell response to antigens. These fusion proteins can bepurchased from commercial vendors such as, for example, R & D Systems(Minneapolis, Minn., USA), or can be isolated as described in Example 5for the BTL-II:Fc protein. B7-2 was purchased from R & D Systems, andB7RP-1 was made essentially as described in Example 5, although it alsois available from R & D Systems under the name B7-H2/Fc. The microtiterplate wells were coated by adding 100 μl of phosphate buffered saline(PBS) containing the concentration of anti-CDR antibody indicated inFIG. 12 with or without one or more other proteins, that is BTL-II:Fc(10 μg/ml), B7RP1:Fc (10 μg/ml), and/or B7-2:Fc (2 μg/ml). Plates wereincubated at 4° C. overnight and then washed twice with PBS. Human Tcells were purified from human peripheral blood mononuclear cells usinga CD4⁺ T cell isolation kit from Miltenyi Biotec (Bergisch Gladbach,Germany), which functions by magnetically labeling and depletingperipheral blood cells other than CD4⁺ T cells, resulting in relativelypure population of untouched CD4⁺ T cells. About 1×10⁵ purified T cellswere added to each well in a volume of 200 μl of culture medium (RPMIwith 10% fetal bovine serum). The cells were incubated for a total of 72hours. At 64 hours, 1 μCi of ³H-thymidine was added to each well. At theend of the 72 hours, unincorporated thymidine was removed by using anautomatic cell harvester (obtained from Tomtec, Hamden, Conn., USA),which deposits the cells onto a filter and washes away the culturemedium. The filters with the cells on them were then counted in ascintillation counter to determine how much radioactivity the cells hadincorporated. The results are shown in FIGS. 12 a and 12 b, which differonly in that FIG. 12 a has a logarithmic scale and FIG. 12 b has alinear scale.

The results indicate that BTL-II:Fc can suppress the T cellproliferation induced by anti-CD3ε antibody. FIGS. 12 a and 12 b. InFIG. 12 a, samples containing BTL-II plus anti-CD3ε antibody (

) proliferate less than samples containing only anti-CD3ε antibody (

) (at the higher antibody concentrations tested. Moreover, it is clearthat both B7-2 (

) and B7RP-1 (

) stimulate anti-CD3ε antibody-induced T cell proliferation. Theconcentration of anti-CD3ε antibody required to see an effect on T cellproliferation by adding B7-2:Fc is lower than that required to see asimilar effect by adding B7RP-1:Fc. Further, BTL-II can also suppressthis increased T cell proliferation in response to the addition ofB7-2:Fc (

) and B7RP-1:Fc (

). The effect of BTL-II:Fc on proliferation induced by B7-2:Fc plusanti-CD3ε antibody is evident only at the lowest concentration ofanti-CD3ε antibody tested, whereas the effects of BTL-II:Fc onproliferation induced by B7RP-1:Fc are more apparent at the three higherconcentrations of anti-CD3ε antibody tested. Thus, the observed effectsdepend on the concentration of anti-CD3ε antibody

FIG. 13 shows that the suppression of human T cell proliferation byBTL-II:Fc is also dependent on BTL-II:Fc concentration. The experimentwas performed as described above except that the anti-CD3ε antibodyconcentration remained constant at 0.5 μg/ml. The BTL-II:Fcconcentration varied from 0 to 10 μg/ml, as indicated in FIG. 13. Theresults shown in FIG. 13 indicate that suppression of anti-CD3ε-inducedT cell proliferation is dependent on the concentration of BTL-II:Fc.

Example 7 BTL-II:Fc Suppresses Murine T Cell Proliferation

This experiment was done to determine whether BTL-II:Fc can suppress theproliferation of murine, as well as human, T cells. A T cellproliferation assay was performed as essentially described in Example 6except that murine T cells purified using magnetic microbeads purchasedfrom Miltneyi Biotec GmbH (Bergisch Gladbach, Germany) were used insteadof human T cells. A protein consisting of a human Fc region, which wasmade in transfected CHO cells, served as a control. The wells werecoated with either anti-CD3ε antibody alone (

) at the concentration indicated in FIG. 14 or with anti-CD368 antibodyplus either BTL-II:Fc (

) at 10 μg/ml or the protein consisting of a human Fc region (

) at 10 μg/ml. The results indicate that BTL-II:Fc, but not a proteinconsisting of a human Fc region alone, can suppress murine T cellproliferation in response to anti-CD3ε antibody. FIG. 14.

Example 8 BTL-II:Fc Does Not Supress Murine B Cell Proliferation

The following experiment was designed to determine whether humanBTL-II:Fc can inhibit proliferation of murine B cells induced by TALL-1and an IgM antibody. The experiment was performed essentially asdescribed by Khare et al. ((2000), Proc. Natl. Acad. Sci. 97(7):3370-75). Briefly, murine B cells were purified from spleens by firstpurifying lymphocytes by density gradient centrifugation and thenpassing the lymphocytes over a B cell column, which removesmonocytes/macrophages and CD4⁺ and CD8⁺ cells (Cedarlane, Westbury,N.Y.). About 1×10⁵ purified B cells in MEM plus 10% heat inactivatedfetal calf serum were incubated for 4 days at 37° C. in a 96 wellmicrotiter plate with or without goat F(ab′)₂ anti-mouse IgM (2 μg/ml),human TALL-1 (10 ng/ml), and/or BTL-II:Fc (10 μg/ml). ³H-thymidine (1μCi) was added during the last 8 hours of incubation. Cells wereharvested as described in Example 6 at the end of 4 days and counted ina scintillation counter. The markings in FIG. 15 indicate the followingcombinations of cells and proteins: B cells alone,

(This is the leftmost bar on the graph shown in FIG. 15, but there is nodetectable signal); B cells plus TALL-1,

; B cells plus anti-IgM antibody,

; B cells plus TALL-1 and anti-IgM antibody,

; and B cells plus TALL-1, anti-IgM antibody, and BTL-II: Fc,

. The results indicate that BTL-II:Fc has no effect on the proliferationof B cells induced by TALL-1 and anti-IgM antibody. FIG. 15. Therefore,BTL-II appears to inhibit proliferation of T cells, but not of B cells.

Example 9 Effects of BTL-II:Fc on Cytokine Production by T Cells

The following experiment was aimed at determining whether BTL-II:Fc haseffects on cytokine production by T cells. T cells were purified andmicrotiter plate wells were coated with proteins as described in Example6. About 1×10⁵ cells were added to each well in a volume of 200 μl ofmedium (RPMI with 10% fetal bovine serum). Cells were incubated for 64hours at 37° C. Then 150 μl was removed to determine cytokineconcentration, and 1 μi of ³H-thymidine was added to the remaining 50 μlin each well. The microtiter plate was then allowed to incubate for anadditional 8 hours at 37° C. and then washed and counted as described inExample 6 to ascertain differences in proliferation (shown in FIG. 16a). Markings to indicate what proteins were used to coat the well are asin FIGS. 12 a and 12 b (explained in Example 6). Concentrations ofinterferon gamma (IFNγ), interleukin 2 (IL2), and interleukin 5 (IL5)were determined using an electrochemical-based immunoassay system forsimultaneous detection of multiple cytokines sold by Meso ScaleDiscovery (MSD, Gaithersburg, Md., USA, which affiliated with IGENInternational, Inc.). The principles and operation of this kind ofcytokine detection system are explained, in e.g., Sennikov et al.(2003), J. Immunol. Methods 275: 81-88. The units for the amounts ofcytokines are taken from the readings generated by the MSD machine. Theactual concentrations of cytokines in the medium cannot be determinedfrom this data without comparison to a standard curve generated with aprotein of known concentration, which did not accompany this particularexperiment. However, comparisons between readings within a singleexperiment can provide relative amounts of cytokines present in controlversus experimental samples.

The results are shown in FIGS. 16 a-16 d. FIG. 16 a indicates thatBTL-II:Fc inhibited proliferation in response to anti-CD3ε antibody oranti-CD3ε antibody plus B7RP-1:Fc, but not in response to anti-CD3εantibody plus B7-2:Fc at the concentrations of anti-CD3ε antibody used.However, as noted in Example 6 (FIGS. 12 a and 12 b), at lower anti-CD3εantibody concentrations, i.e., 0.1 μg/ml, cell proliferation in responseto anti-CD3ε antibody plus B7-2:Fc is inhibited by BTL-II:Fc. Productionof IFNγ, IL2, and IL5 was low in wells coated with anti-CD3ε antibodyalone, and addition of BTL-II:Fc did not decrease it significantlyfurther. FIGS. 16 b-16 d. Increased IFNγ production in response to theaddition of either B7RP-1:Fc or B7-2:Fc to anti-CD3ε antibody wasinhibited by BTL-II:Fc. FIG. 16 b. In addition, increased IL2 and IL5production in response to the addition of B7-2:Fc to anti-CDR antibodywas inhibited by BTL-II:Fc. FIGS. 16 c and 16 d. At the concentrationstested, the addition of B7RP-1:Fc to anti-CD3ε antibody did notappreciably increase IL2 or IL5 production. FIGS. 16 c and 16 d. Theseresults indicate that BTL-II:Fc can inhibit production of at least somecytokines in response to a combination of anti-CDR antibody plus eitherB7-2:Fc or B7RP-1:Fc.

Example 10 Treatment of T Cells with BTL-II:Fc Does Not Result inMassive Cell Death

The following experiment was done to determine whether inhibition of Tcell proliferation by BTL-II:Fc involved massive cell death. A T cellproliferation assay was performed essentially as described in Example 6,except that the cells were not labeled with ³H-thymidine. Cells werecounted in a hemocytometer after 72 hours of culture, and cell viabilitywas determined by trypan blue staining Proteins used to coat themicrotiter plate wells are indicated in FIG. 17 as explained in Example6 and shown in FIGS. 12 a and 12 b. The anti-CD3ε antibody was used at aconcentration of 2 μg/ml. The results indicate that the number of deadcells in each well falls within a range between about 0.5×10⁴ and0.75×10⁴ cells, regardless of the proteins used to coat the wells. FIG.17. Therefore, the differences in cell proliferation observed in thepresence of BTL-II:Fc do not reflect a substantial toxic effect ofBTL-II:Fc.

Example 11 Murine BTL-II Does Not Bind to Murine CTLA4, CD28, ICOS, orPD-1

The following set of experiments addresses the question of whetherBTL-II binds to one of several known binding partners of other B7proteins. For example, B7RP-1 is known to bind to ICOS (Yoshinaga et al.(1999), Nature 402: 827-32), CD80 (also called B7-1) is known to bind toCTLA4 and CD28, as does CD86 (also called B7-2; see e.g. Sharpe andFreeman (2002), Nature Reviews Immunology 2: 116-26), and PD-L1 andPD-L2 are known to bind to PD-1 (Latchman et al. (2001), NatureImmunology 2(3): 261-68).

The experiment was done as follows. First, fusion proteins comprisingthe extracellular region of a protein known to bind a B7 protein plusthe Fc region of a human IgG antibody were obtained or isolated. Fusionproteins comprising an Fc region of a human antibody plus theextracellular region of either murine CD28 (mCD28-huFC) or murine PD-1(PD1-huFC) were purchased from R & D Systems (Minneapolis, Minn., USA).The other two B7 binding proteins (CTLA4-huFC and ICOS-huFC) are alsoavailable from R & D Systems, but were made as follows. A cDNA encodingthe extracellular region of murine CTLA4 and another encoding theextracellular region of murine ICOS were each fused to cDNA encoding theFc region of a human IgG antibody in a vector appropriate for expressionin mammalian cells. Each of these constructs was used to transfectcells, and the fusion proteins were purified from the culture medium ofthe transfected cells by Protein A chromatography.

Full length versions of each of three murine cDNAs (encoding BTL-II,B7RP-1, CD80) were inserted into a vector appropriate for expression.About 10 μg of each of these constructs, along with an empty vector,were used separately to transfect 293 cells. Two days post transfection,about one million cells from each of the four transfections were stainedwith each of the fusion proteins described above. Bound protein wasdetected using a fluorescently labeled antibody against the human IgG Fcregion. The stained cells were analyzed by FACS. The results are shownin FIG. 18.

As expected, cells transfected with the empty vector (top line of FIG.18) did not stain with any of the four fusion proteins. Cellstransfected with murine BTL-II (second line of FIG. 18) behavedsimilarly, indicating that none of the fusion proteins bind to BTL-II.As expected, cells transfected with murine B7RP-1 stained withICOS-huFC, but not with any of the other fusion proteins. Also asexpected, cells transfected with murine CD80 stained with CTLA4-huFC ormCD28-huFC, but not with ICOS-huFC or PD1-huFC. These results indicatethat BTL-II fails to bind to four proteins, CTLA-4, PD-1, ICOS, andCD28, each of which is known to bind to at least one protein in the B7family.

1. A method for inhibiting T cell proliferation and/or cytokineproduction by a T cell comprising contacting a T cell with a BTL-IIprotein, whereby proliferation of the T cell and/or cytokine productionby the T cell is inhibited, and wherein the BTL-II protein is selectedfrom the group consisting of: (a) a BTL-II protein comprising the aminoacid sequence of amino acids 29 to 457 of SEQ ID NO:4; (b) a BTL-IIprotein comprising an amino acid sequence at least 85% identical toamino acids 30 to 358 of SEQ ID NO:10, wherein the identity region ofthe amino acid sequence aligned with amino acids 30 to 358 of SEQ IDNO:10, is at least 150 amino acids, wherein the amino acid sequence isat least 85% identical to amino acids 127 to 157 of SEQ ID NO:10,wherein the identity region of the amino acid sequence aligned withamino acids 127 to 157 of SEQ ID NO:10, is at least 20 amino acids long,and wherein the BTL-II protein can inhibit the proliferation of T cellsinduced by an anti-CD3 antibody; (c) a BTL-II protein comprising anamino acid sequence at least 85% identical to amino acids 32 to 358 ofSEQ ID NO:10, wherein the identity region of the amino acid sequencealigned with amino acids 32 to 358 of SEQ ID NO:10 is at least 275 aminoacids, and wherein the BTL-II protein can inhibit the proliferation of Tcells induced by an anti-CD3 antibody; or (d) a BTL-II proteincomprising a first amino acid sequence at least 85% identical to aminoacids 32 to 358 of SEQ ID NO:10, wherein the identity region of thefirst amino acid sequence aligned to SEQ ID NO:10 is at least about 175amino acids long, wherein the first amino acid sequence is not more than390 amino acids in length, wherein the BTL-II protein can inhibit theproliferation of T cells induced by an anti-CD3 antibody, wherein thefirst amino acid sequence is not at least 85% identical to amino acids148 to 232 of SEQ ID NO:4 with an identity region of the first aminoacid sequence aligned to amino acids 148 to 232 of SEQ ID NO:4 of atleast about 40 amino acids, and wherein the protein does not comprise asecond amino acid sequence that is at least 85% identical to amino acids148 to 232 of SEQ ID NO:4 with an identity region of the second aminoacid sequence aligned to amino acids 148 to 232 of SEQ ID NO:4 of atleast about 40 amino acids.
 2. The method of claim 1, wherein the BTL-IIprotein is the BTL-II protein described in part (a) of claim
 1. 3. Themethod of claim 1, wherein the BTL-II protein is the BTL-II proteindescribed in part (b) of claim
 1. 4. The method of claim 1, wherein theBTL-II protein is the BTL-II protein described in part (c) of claim 1.5. The method of claim 1, wherein the BTL-II protein is the BTL-IIprotein described in part (d) of claim
 1. 6. The method of claim 1,wherein the BTL-II protein further comprises an additional amino acidsequence.
 7. The method of claim 6, wherein the additional amino acidsequence is an Fc region of an antibody.
 8. The method of claim 1,wherein the T cell is a human T cell.
 9. The method of claim 1, wherebycytokine production by the T cell is inhibited.
 10. The method of claim9, whereby the production of interferon gamma, IL2 and/or IL5 by the Tcell is inhibited.
 11. The method of claim 1, whereby proliferation ofthe T cell is inhibited.
 12. A method for treating an inflammatory boweldisease comprising administering to a patient suffering from theinflammatory bowel disease a therapeutically effective amount of aBTL-II protein, whereby the disease is treated, and wherein the BTL-IIprotein is selected from the group consisting of: (a) a BTL-II proteincomprising the amino acid sequence of amino acids 29 to 457 of SEQ IDNO:4; (b) a BTL-II protein comprising an amino acid sequence at least85% identical to amino acids 30 to 358 of SEQ ID NO:10, wherein theidentity region of the amino acid sequence aligned with amino acids 30to 358 of SEQ ID NO:10, is at least 150 amino acids, wherein the aminoacid sequence is at least 85% identical to amino acids 127 to 157 of SEQID NO:10, wherein the identity region of the amino acid sequence alignedwith amino acids 127 to 157 of SEQ ID NO:10, is at least 20 amino acidslong, and wherein the BTL-II protein can inhibit the proliferation of Tcells induced by an anti-CD3 antibody; (c) a BTL-II protein comprisingan amino acid sequence at least 85% identical to amino acids 32 to 358of SEQ ID NO:10, wherein the identity region of the amino acid sequencealigned with amino acids 32 to 358 of SEQ ID NO:10 is at least 275 aminoacids, and wherein the BTL-II protein can inhibit the proliferation of Tcells induced by an anti-CD3 antibody; or (d) a BTL-II proteincomprising a first amino acid sequence at least 85% identical to aminoacids 32 to 358 of SEQ ID NO:10, wherein the identity region of thefirst amino acid sequence aligned to SEQ ID NO:10 is at least about 175amino acids long, wherein the first amino acid sequence is not more than390 amino acids in length, wherein the BTL-II protein can inhibit theproliferation of T cells induced by an anti-CD3 antibody, wherein thefirst amino acid sequence is not at least 85% identical to amino acids148 to 232 of SEQ ID NO:4 with an identity region of the first aminoacid sequence aligned to amino acids 148 to 232 of SEQ ID NO:4 of atleast about 40 amino acids, and wherein the protein does not comprise asecond amino acid sequence that is at least 85% identical to amino acids148 to 232 of SEQ ID NO:4 with an identity region of the second aminoacid sequence aligned to amino acids 148 to 232 of SEQ ID NO:4 of atleast about 40 amino acids.
 13. The method of claim 12, wherein theinflammatory bowel disease is Crohn's disease.
 14. The method of claim12, wherein the inflammatory bowel disease is ulcerative colitis. 15.The method of claim 12, wherein the BTL-II protein is the BTL-II proteindescribed in part (a) of claim
 12. 16. The method of claim 12, whereinthe BTL-II protein is the BTL-II protein described in part (b) of claim12.
 17. The method of claim 12, wherein the BTL-II protein is the BTL-IIprotein described in part (c) of claim
 12. 18. The method of claim 12,wherein the BTL-II protein is the BTL-II protein described in part (d)of claim
 12. 19. The method of claim 12, wherein the BTL-II proteinfurther comprises an additional amino acid sequence.
 20. The method ofclaim 19, wherein the additional amino acid sequence is an Fc region ofan antibody.